Luciferase biosensor

ABSTRACT

A modified beetle luciferase protein which is an environmentally sensitive reporter protein is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/957,433, filed Oct. 1, 2004, which claims priority to U.S.Provisional Application No. 60/510,187, filed Oct. 10, 2003, thedisclosures of which are both incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to the field of biochemical assays and reagents.More specifically, this invention relates to modified reporter proteins,e.g., luminescent reporter proteins, and to methods for their use.

BACKGROUND

Luciferases are enzymes that catalyze the oxidation of a substrate(e.g., luciferin) with the concomitant release of photons of light.Luciferases have been isolated from numerous species, includingColeopteran arthropods and many sea creatures. Because it is easilydetectable and its activity can be quantified with high precision,luciferase/substrate pairs have been used widely to study geneexpression and protein localization. Unlike another reporter protein,green fluorescent protein (GFP), which requires up to 30 minutes to formchromophore, the products of luciferases can be detected immediatelyupon completion of synthesis of the polypeptide chain (if substrate andoxygen are also present). In addition, no post-translationalmodifications are required for enzymatic activity, and the enzymecontains no prosthetic groups, bound cofactors, or disulfide bonds.Luciferase is a useful reporter in numerous species and in a widevariety of cells.

Luciferases possess additional features that render them particularlyuseful as reporter molecule's for biosensing, i.e., molecules whichreveal properties of a biological system. Signal transduction inbiosensors (sensors which comprise a biological component) generallyinvolves a two-step process: signal generation through a biologicalcomponent, and signal transduction and amplification through anelectrical component. Signal generation is typically achieved throughbinding or catalysis. Conversion of these biochemical events into anelectrical signal is typically based on electrochemical or caloricdetection methods, which are limited by the free energy change of thebiochemical reactions. For most reactions, this is less than the energyof hydrolysis for two molecules of ATP, or about 70 kJ/mole. However,the luminescence elicited by luciferases has a much higher energycontent. For instance, the reaction catalyzed by firefly luciferase (560nm) emits 214 kJ/mole of energy. Furthermore, the reaction catalyzed byluciferase is one of the most efficient bioluminescent reactions known,having a quantum yield of nearly 0.9. Luciferase is thus an extremelyefficient transducer of chemical energy.

Luciferase biosensors have been described. For example, Sala-Newby etal. (1991) disclose that a Photinus pyralis luciferase cDNA wasamplified in vitro to generate cyclic AMP-dependent protein kinasephosphorylation sites. In particular, a valine at position 217 wasmutated to arginine to generate a site, RRFS, and the heptapeptidekemptide, the phosphorylation site of the porcine pyruvate kinase, wasadded at the N- or C-terminus of the luciferase. Sala-Newby et al.relate that the proteins carrying phosphorylation sites werecharacterized for their specific activity, pI, effect of pH on the colorof the light emitted, and effect of the catalytic subunit of proteinkinase A in the presence of ATP. They found that only one of therecombinant proteins (RRFS) was significantly different from wild-typeluciferase and that the RRFS mutant had a lower specific activity, lowerpH optimum, emitted greener light at low pH and, when phosphorylated,decreased its activity by up to 80%. It is disclosed that the lattereffect was reversed by phosphatase.

Waud et al. (1996) engineered protein kinase recognition sequences andproteinase sites into a Photinus pyralis luciferase cDNA. Two domains ofthe luciferase were modified by Waud et al.; one between amino acids 209and 227 and the other at the C-terminus, between amino acids 537 and550. Waud et al. disclose that the mutation of amino acids betweenresidues 209 and 227 reduced bioluminescent activity to less than 1% ofwild-type recombinant, while engineering peptide sequences at theC-terminus resulted in specific activities ranging from 0.06%-120% ofthe wild-type recombinant luciferase. Waud et al. also disclose thataddition of a cyclic AMP dependent protein kinase catalytic subunit to avariant luciferase incorporating the kinase recognition sequence,LRRASLG (SEQ ID NO:107), with a serine at amino acid position 543,resulted in a 30% reduction activity. Alkaline phosphatase treatmentrestored activity. Waud et al. further disclose that the bioluminescentactivity of a variant luciferase containing a thrombin recognitionsequence, LVPRES (SEQ ID NO: 108), with the cleavage site positionedbetween amino acids 542 and 543, decreased by 50% when incubated in thepresence of thrombin.

Ozawa et al. (2001) describe a biosensor based on proteinsplicing-induced complementation of rationally designed fragments offirefly luciferase. Protein splicing is a posttranslational proteinmodification through which inteins (internal proteins) are excised outfrom a precursor fusion protein, ligating the flanking exteins (externalproteins) into a contiguous polypeptide. It is disclosed that the N- andC-terminal intein DnaE from Synechocystis sp. PCC6803 were each fusedrespectively to N- and C-terminal fragments of a luciferase.Protein-protein interactions trigger the folding of DnaE intein,resulting in protein splicing, and thereby the extein of ligatedluciferase recovers its enzymatic activity. Ozawa et al. disclose thatthe interaction between known binding partners, phosphorylated insulinreceptor substrate 1 (IRS-1) and its target N-terminal SH2 domain of PI3-kinase, was monitored using a split luciferase in the presenceinsulin.

Paulmurugan et al. (2002) employed a split firefly luciferase-basedassay to monitor the interaction of two proteins, i.e., MyoD and Id, incell cultures and in mice using both complementation strategy and anintein-mediated reconstitution strategy. To retain reporter activity, inthe complementation strategy, fusion proteins need protein interaction,i.e., via the interaction of the protein partners MyoD and Id, while inthe reconstitution strategy, the new complete reporter protein formedvia intein-mediated splicing maintains it activity even in the absenceof a continuing interaction between the protein partners.

A protein fragment complementation assay is disclosed in Michnick et al.(U.S. Pat. Nos. 6,270,964, 6,294,330 and 6,428,951). Specifically,Michnick describe a split murine dihydrofolate reductase (DHFR)gene-based assay in which an N-terminal fragment of DHFR and aC-terminal fragment of DHFR are each fused to a GCN4 leucine zippersequence. DHFR activity was detected in cells which expressed bothfusion proteins. Michnick et al. also describe another complementationapproach in which nested sets of S1 nuclease generated deletions in theaminoglycoside kinase (AK) gene are introduced into a leucine zipperconstruct, and the resulting sets of constructs introduced to cells andscreened for AK activity.

What is needed is an improved recombinant reporter protein for use as abiosensor, e.g., in detecting cellular events such as protein-proteininteractions, with a high degree of specificity and a high quantumyield.

SUMMARY OF THE INVENTION

The invention provides an improved gene product, e.g., a modifiedreporter protein such as a modified beetle luciferase, which, in thepresence of another molecule (one or more molecules of interest), orunder certain conditions, has one or more altered activities. In oneembodiment, the amino acid sequence of the modified reporter protein isdifferent than the amino acid sequence of a corresponding unmodified(native, wild-type or parental) reporter protein as a result of one ormore modifications at a site (residue) or in a region which is tolerantto modification, e.g., tolerant to an insertion, a deletion, circularpermutation, or any combination thereof. One or more modifications maybe internal to the N- or C-terminus of the unmodified reporter protein,and/or may be at the N- and/or C-terminus of the unmodified reporterprotein, e.g., a deletion and/or insertion of one or more amino acidresidues, thereby yielding a modified reporter protein. Themodification(s) may include the introduction of one or more discreet(isolated) amino acid sequences which directly or indirectly interactwith a molecule of interest and/or is/are otherwise sensitive to changesin conditions, and optionally may include the deletion of one or moreamino acids, e.g., at a site or in a region tolerant to modificationincluding the N- and/or C-terminus of the unmodified reporter protein,so long as the resulting modified reporter protein has reporter activitybefore and/or after the interaction with the molecule of interest, suchas an exogenous agent, or a change in conditions. For instance, themodified reporter protein may include deletions at the N- or C-terminusof 1 to about 10 or 15 residues, or any integer in between, relative tothe corresponding unmodified reporter protein. The modification may bethe absence of a peptide bond in the modified reporter protein betweentwo amino acids which are linked via a peptide bond in the correspondingunmodified reporter protein, in conjunction with a peptide bond in themodified reporter protein between residues found at or near theN-terminal and C-terminal residues of the corresponding unmodifiedreporter protein, yielding a circularly permuted reporter protein, whichoptionally includes an amino acid sequence which directly or indirectlyinteracts with a molecule of interest or is otherwise sensitive tochanges in conditions. The modified reporter protein may thus beemployed to detect reversible interactions, e.g., binding of two or moremolecules, formation of disulfide bonds or other conformational changesor changes in conditions, such as pH, temperature or solventhydrophobicity, or irreversible interactions, e.g., cleavage of apeptide bond, via an alteration in the activity of the modified reporterprotein, such as an alteration in light intensity, color or kineticprofile.

As described hereinbelow, Tn5 was employed to prepare a library ofinsertions of DNA encoding 19 amino acids into a click beetle luciferasenucleic acid sequence. Analysis of 416 clones with insertions showedthat about 10% (52) of the clones had partial activity, e.g., activitiesup to 2% of wild-type. Of the 52 clones, 27 clones had insertions in theluciferase open reading frame, and 16 of those insertions were betweenresidues 398 to 409 (the “hinge” region). In particular, in-frameinsertions resulting in modified click beetle luciferases withdetectable activity were at residue 21, 25, 117, 358, 376, 379, 398,399, 400, 401, 402, 403, 405, 406, 407, 409 or 490 of click beetleluciferase, i.e., those residues and/or regions near those residues aretolerant to modification including insertions. Thus, the inventionincludes a modified beetle luciferase with a modification at a residue,for instance residue 21, 25, 117, 358, 376, 379, 398, 399, 400, 401,402, 403, 405, 406, 407, 409 or 490, or in a region corresponding toresidue 15 to 30, e.g., residue 21 or 25, residue 112 to 122, e.g.,residue 117, residue 352 to 362, for instance, residue 358, residue 371to 384, e.g., residue 379, residue 393 to 414, or residue 485 to 495, ofa click beetle luciferase. Corresponding positions may be identified byaligning luciferase sequences. In particular, the invention includes amodified beetle luciferase with a modification in the hinge region ofbeetle luciferase, e.g., residues corresponding to residues 390 to 409of click beetle luciferase, as well as other regions which can toleratemodification.

As also described herein, Tn7 was employed to prepare a library ofinsertions into a firefly luciferase nucleic acid sequence. In-frameinsertions resulting in modified firefly luciferases with detectableactivity were at residue 7, 121, 233, 267, 294, 303, 361, 540 or 541 offirefly luciferase, i.e., those residues and/or regions near thoseresidues are tolerant to modifications including insertions.Accordingly, the invention includes a modified beetle luciferase with amodification at a residue or in a region corresponding to residue 2 to12, residue 116 to 126, residue 228 to 238, residue 262 to 272, residue289 to 308, residue 356 to 366, or residue 535 to 546, of a fireflyluciferase. Corresponding positions may be identified by aligningluciferase sequences.

Thus, in one embodiment, the reporter protein is a beetle luciferase,and the amino acid sequence of the modified beetle luciferase isdifferent than the amino acid sequence of a corresponding unmodifiedbeetle luciferase as a result of one or more modifications at a site orin a region which is tolerant to modification. For example, in oneembodiment, the modified beetle luciferase has a detectable activity andincludes an insertion of one or more amino acids relative to acorresponding unmodified beetle luciferase at a site or in a regionwhich is tolerant to modification, which insertion is internal to the N-and C-terminus of the modified beetle luciferase. In one embodiment, amodified beetle luciferase comprises an insertion of 2 or more, e.g., 3,4, 5, 10, 20, 50, 100, 200, 300 or more, but less than about 500, or anyinteger in between, amino acid residues. In one embodiment, a modifiedbeetle luciferase of the invention comprises an internal insertion of atleast 4 amino acids at a residue or in a region which is tolerant tomodification, which insertion includes an amino acid sequence whichdirectly interacts with a molecule of interest, e.g., an insertion whichincludes a recognition sequence for the molecule of interest, orindirectly acts with the molecule of interest, e.g., via anothermolecule. In one embodiment, the modified beetle luciferase with aninternal insertion further comprises an internal deletion of beetleluciferase sequences, e.g., a deletion of 1 or more, but less than about100, for instance less than 50, 40, 30, 20, 10 or 5, or any integer inbetween, residues.

In one embodiment, the modified beetle luciferase has a deletionrelative to a corresponding unmodified beetle luciferase, at a site orin a region which is tolerant to modification. In one embodiment, amodified beetle luciferase of the invention comprises a deletion of atleast 50, e.g., at least 100, contiguous amino acid residues relative toa corresponding unmodified beetle luciferase, i.e., the modified beetleluciferase is a fragment of a full-length unmodified beetle luciferasesequence, e.g., a fragment of at least 50, e.g., at least 100,contiguous amino acid residues, for instance, a fragment which has atleast 5%, e.g., 10%, fewer residues than the corresponding full-lengthunmodified beetle luciferase, and an insertion of an amino acid sequencewhich directly or indirectly interacts with a molecule of interest or isotherwise sensitive to conditions. Such a modified beetle luciferase maybe employed in a protein complementation assay, e.g., where a detectableactivity of the luciferase increases in the presence of another fragmentof the luciferase which is linked to a molecule of interest, or in aprotein recombination assay, for instance, intein-mediatedrecombination. In one embodiment, a beetle luciferase fragment (withoutone or more heterologous sequences) has a detectable activity which isless than, e.g., about 0.001%, 0.01%, 0.1% or 1%, the activity of thecorresponding full-length unmodified beetle luciferase and, whencombined with a complementing fragment (without one or more heterologoussequences), has an increase in activity relative to either fragment ofgreater than 3-fold, e.g., 10-, or 50- to 100-fold or more. Forinstance, in one embodiment, the N-terminal beetle luciferase fragmenthas at least 0.001% but less than 1%, and the C-terminal beetleluciferase fragment has at least 0.01% but less than 5%, the activity ofthe corresponding full-length unmodified beetle luciferase. In anotherembodiment, a modified beetle luciferase of the invention is a fragmentwhich has a deletion of at least 50, e.g., at least 100, contiguousamino acid residues relative to a corresponding unmodified beetleluciferase, an insertion of an amino acid sequence which directly orindirectly interacts with a molecule of interest or is otherwisesensitive to conditions, and an insertion of heterologous, e.g.,non-beetle luciferase, sequences, which insertions preferably do notincrease but may individually or together decrease the activity of thebeetle luciferase fragment, but which, once removed, result in atruncated beetle luciferase with increased activity relative to themodified beetle luciferase.

As further described herein, circularly permuted firefly and clickbeetle luciferases, having a N-terminus at a residue or in a regionwhich is tolerant to modification in the corresponding noncircularlypermuted beetle luciferase, and optionally including an amino acidsequence which directly or indirectly interacts with a molecule ofinterest, e.g., a protease recognition site or a kinase site, wereprepared and shown to have detectable activity, which activity wasaltered in the presence of the molecule of interest, for instance, asuitable protease or kinase in constructs which encoded a proteaserecognition site or a kinase site, respectively, in the circularlypermuted luciferase. Hence, in one embodiment, a modified beetleluciferase of the invention comprises an amino acid sequence which iscircularly permuted relative to the amino acid sequence of acorresponding unmodified beetle luciferase, resulting in a new N- andC-terminus in the modified beetle luciferase, at least one of which isat a site or in a region which is tolerant to modification. In anotherembodiment, the circularly permuted beetle luciferase includes othermodifications, including but not limited to insertions and/or deletionsinternal to the N- or C-terminus of the circularly permuted beetleluciferase, for instance, an insertion and/or deletion, e.g., at or nearthe N- and C-terminus of the corresponding unmodified beetle luciferasesuch as at residues corresponding to residues 1 to about 10 or 15, orany integer in between, of the N-terminus and/or corresponding to thelast residue or about the last 15, or any integer in between 1 and 15,residues of the C-terminus of the corresponding unmodified beetleluciferase. Thus, the N- and C-termini of a reporter protein can bealtered via circular permutation, and the resulting permuted moleculemay have one or more activities of the nonpermuted reporter protein.Accordingly, a circularly permuted reporter protein may be employed in aprotein complementation assay or in a protein recombination assay.Moreover, a circularly permuted reporter protein may be engineered tohave functionality by introducing an amino acid sequence which directlyor indirectly interacts with a molecule of interest or is otherwisesensitive to changes in conditions. In one embodiment, a circularlypermuted reporter protein of the invention is a zymogen.

In one embodiment, in the absence of the molecule of interest, theactivity of a modified reporter protein such as a modified beetleluciferase is less than the activity of a corresponding unmodifiedreporter protein, e.g., the reporter activity of the modified beetleluciferase is about 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 50%, 70% or more,but less than 100% that of a corresponding unmodified beetle luciferase,the activity of which modified reporter protein is optionallydetectable. In another embodiment, in the absence of the molecule ofinterest, the activity of a modified reporter protein such as a modifiedbeetle luciferase is greater than the activity of a correspondingunmodified reporter protein, e.g., the reporter activity of the modifiedbeetle luciferase is about 1.5-fold, e.g., at least 2-, 3- or 5-fold ormore, that of a corresponding unmodified beetle luciferase. In thepresence of the molecule of interest, the activity of the modifiedreporter protein is detectably altered. For instance, a detectablealteration in activity of a modified beetle luciferase in the presenceof a molecule of interest is an alteration of at least 0.001%, 0.01%,0.1%, 1%, 10%, or 100%, and up to 2-fold, 4-fold, 10-fold, 100-fold,1,000-fold, 10,000-fold or more, relative to the activity of themodified beetle luciferase in the absence of the molecule of interest.Thus, the physical proximity of a molecule of interest which interactswith a modification present in the modified reporter protein but not thecorresponding unmodified reporter protein, alters, e.g., decreases,eliminates or increases, the activity of the modified reporter protein.For example, a modified beetle luciferase may comprise an internalinsertion relative to a corresponding unmodified beetle luciferase,which insertion comprises a protease recognition site, i.e., a sitewhich is cleaved by a protease. The luminescent signal of such amodified beetle luciferase in the presence of the protease may bedecreased, eliminated or increased relative to the luminescent signal ofthe modified beetle luciferase in the absence of the protease or theluminescent signal of the corresponding unmodified beetle luciferase inthe presence or absence of the molecule of interest. Alternatively, amodified beetle luciferase which comprises a deletion relative to acorresponding unmodified beetle luciferase, may be fused to a ligandwhich interacts with a molecule of interest. A complementing secondfragment of a beetle luciferase is fused to the molecule of interest andthe two fusions are allowed to interact, an interaction which alters,e.g., increases, the activity of the resulting complex relative to theactivity of either fusion alone. In one embodiment, one fragment of abeetle luciferase has residues corresponding to residues about 1 to 126,about 1 to about 238, about 1 to about 272, about 1 to about 308, about1 to about 366, about 116 to about 550, about 228 to about 550, about262 to about 550, about 289 to about 550, or about 356 to about 550, orany integer in between, of a firefly luciferase, or residues about 1 toabout 122, about 1 to about 362, about 1 to about 384, about 1 to about414, about 352 to about 542, about 371 to about 542, or about 393 toabout 542, or any integer in between, of a click beetle luciferase.

The invention also provides for a modified reporter protein whichincludes heterologous sequences at the N-terminus and C-terminus of areporter protein, i.e., the modified protein is a fusion protein, whichheterologous sequences noncovalently interact, that is, the twoheterologous sequences are binding partners. In one embodiment, themodified reporter protein is a circularly permuted beetle luciferasewhich includes heterologous sequences at the N-terminus and C-terminus.In one embodiment, in the absence of one or more exogenous agents (atleast one of which may be a molecule of interest, e.g., one which is tobe detected or identified in a sample), a modified reporter proteinwhich has both heterologous sequences, one at the N-terminus and theother at the C-terminus, has less, the same or greater activity than acorresponding unmodified reporter protein. In one embodiment, themodified reporter protein may also lack one or more amino acids presentat the N- and/or C-terminus of the unmodified reporter protein, theabsence of which does not substantially alter the reporter activity ofthe modified reporter protein, e.g., the activity of the reporterportion the modified reporter protein is at least 0.001%, 0.01%, 0.1%,1%, 10%, 50%, 100% or greater than the activity of a correspondingreporter protein without the deletion(s). In one embodiment, in thepresence of one or more exogenous agents or under specified conditions,the activity of the modified reporter protein having both heterologoussequences, but not the corresponding reporter protein without theheterologous sequences (that is the corresponding unmodified reporterprotein), is detectably altered, e.g., by at least 2-, 5-, or 10-fold ormore. For instance, in the presence of rapamycin, a luciferase fused torapamycin binding protein (FRB) and FK506 binding protein (FKBP), hasreduced activity relative to a luciferase which lacks FRB and FKBP. Inone embodiment, in the absence of the exogenous agent(s) or underdifferent conditions, the modified reporter protein does not havedetectable activity, while in other embodiments it has detectableactivity, which activity may be enhanced in the presence of at least oneexogenous agent or under specified conditions. For example, the modifiedreporter protein in the absence of an exogenous agent may have little orno activity, but, after addition of a selected exogenous agent whichenhances the noncovalent interaction of the two heterologous sequences,the activity of the modified reporter protein is enhanced.Alternatively, the activity of the modified reporter protein having bothheterologous sequences may be inhibited in the presence of at least oneexogenous agent or under specified conditions. In one embodiment, oneheterologous sequence includes a domain, e.g., 3 or more amino acidresidues, which optionally may be covalently modified, e.g.,phosphorylated, that noncovalently interacts with a domain in the otherheterologous sequence. Heterologous sequences useful as binding partnerswhen fused to a beetle luciferase include but are not limited to thosewhich interact in vitro and/or in vivo and optionally which, based onprotein modeling for example, have linked sequences that do notparticipate in binding but are an approximate selected distance apart inthe presence or absence of an exogenous agent which alters theinteraction of the binding partners, such that their fusion to the endsof a beetle luciferase result in a modulatable beetle luciferase.Exemplary heterologous sequences include but are not limited tosequences such as those in FRB and FKBP, the regulatory subunit ofprotein kinase (PKa-R) and the catalytic subunit of protein kinase(PKa-C), a src homology region (SH2) and a sequence capable of beingphosphorylated, e.g., a tyrosine containing sequence, an isoform of14-3-3, e.g., 14-3-3t (see Mils et al., 2000), and a sequence capable ofbeing phosphorylated, a protein having a WW region (a sequence in aprotein which binds proline rich molecules (see Ilsley et al., 2002; andEinbond et al., 1996) and a heterologous sequence capable of beingphosphorylated, e.g., a serine and/or a threonine containing sequence,as well as sequences in dihydrofolate reductase (DHFR) and gyrase B(GyrB).

In another embodiment, in the presence of one (first) exogenous agent, amodified reporter protein which includes heterologous sequences at theN-terminus and C-terminus which are binding partners, has an alteredactivity relative to the activity in the absence of the exogenous agent,and in the presence of a different (second) exogenous agent, theactivity of the modified reporter protein is altered relative to theactivity in the presence of the first exogenous agent, e.g., the secondexogenous agent competes with the first exogenous agent. In oneembodiment, in the absence of the first exogenous agent, the modifiedreporter protein has no or low detectable activity, and the addition ofthe first exogenous agent results in an increase in the activity of themodified reporter protein, which is reversible by the addition of asecond exogenous agent. In another embodiment, in the absence of thefirst exogenous agent, the modified reporter protein has detectableactivity, and the addition of the first exogenous agent results inreduced or a lack of detectable activity, or alternatively an increasein detectable activity, which is reversible by the addition of a secondexogenous agent. The modified reporter protein optionally may lack oneor more amino acids at the N- and/or C-terminus relative to theunmodified reporter protein, for instance a deletion of residue 1 orresidues 1 to about 10 or 15, or any integer in between, of theN-terminus and/or corresponding to the last residue or about the last15, or any integer in between 1 and 15, residues of the C-terminus, ofthe corresponding unmodified reporter protein.

In yet another embodiment, a modified reporter protein includes aheterologous sequence at the N-terminus or C-terminus which heterologoussequence alters, e.g., inhibits, the activity of the modified reporterprotein, which activity is modified, for instance, at least partiallyrestored, by the addition of a first exogenous agent. Optionally, theeffect of the first exogenous agent is reversibly altered by a secondexogenous agent. In one embodiment, the heterologous sequence mayinhibit substrate entry and the conformation of the heterologoussequence is substantially altered in the presence of the first exogenousagent such that the modified reporter protein can interact with itssubstrate. The modified reporter protein optionally may lack one or moreamino acids at the N- and/or C-terminus of the unmodified reporterprotein such as those that correspond to residues 1 to about 10 or 15,or any integer in between, of the N-terminus and/or corresponding to thelast residue or about the last 15, or any integer in between 1 and 15,residues of the C-terminus, of the corresponding unmodified reporterprotein. A heterologous sequence useful in this embodiment is calmodulin(CaM).

Thus, a modified reporter protein may be employed to detect reversibleinteractions of the binding partners, or reversible conformationalchanges of a heterologous sequence, which may be enhanced or inhibitedby one or more agents or changes in conditions, e.g., ionic strength ortemperature.

Accordingly, a modified beetle luciferase of the invention may beemployed as a biosensor.

The invention also provides an isolated nucleic acid molecule(polynucleotide) comprising a nucleic acid sequence encoding a modifiedreporter protein of the invention. Further provided is an isolatednucleic acid molecule comprising a nucleic acid sequence encoding fusionprotein comprising a modified reporter protein and one or more aminoacid residues at the N-terminus (a N-terminal fusion partner) and/orC-terminus (a C-terminal fusion partner) of the modified reporterprotein. Thus, as used herein, a “fusion protein” is a polypeptide whichincludes one or more amino acids at the N-terminus and/or C-terminus ofa modified reporter protein of the invention. Preferably, the presenceof one or more fusion partners in the fusion protein does notsubstantially alter the detectable activity of the fusion proteinrelative to a corresponding modified reporter protein. In oneembodiment, the fusion protein comprises at least two different fusionpartners, one at the N-terminus and another at the C-terminus of amodified reporter protein. The N- or C-terminal fusion partner may be asequence used for purification, e.g., a glutathione S-transferase (GST)or a polyHis sequence, a sequence intended to alter a property of themodified reporter protein, e.g., a protein destabilization sequence or akinase binding domain for a kinase site in the modified reporter proteinat a residue or in a region which is tolerant to modifications, or asequence which has a property which is distinguishable from one or moreproperties of the reporter protein in the fusion protein. In oneembodiment, the fusion protein comprises a modified beetle luciferaseand a fusion partner which is a reporter protein that is different thanthe beetle luciferase, which reporter protein is useful as anintramolecular control, e.g., a fluorescent protein. In anotherembodiment, the invention includes a vector comprising a nucleic acidsequence encoding a fusion protein comprising a modified beetleluciferase of the invention and a nucleic acid fragment which encodes areporter protein that is different than the beetle luciferase in themodified beetle luciferase. Optionally, optimized nucleic acidsequences, e.g., human codon optimized sequences, encoding at least thebeetle luciferase, and preferably the modified beetle luciferase or afusion protein comprising a modified beetle luciferase, are employed inthe nucleic acid molecules of the invention, as those optimizedsequences can increase the strength of the signal for beetle luciferase.The optimization of nucleic acid sequences is known to the art, see, forexample WO 02/16944.

The invention also includes a stable cell line that expresses a modifiedreporter protein, e.g., a beetle luciferase, or fusion protein of theinvention, as well as an expression cassette comprising a nucleic acidmolecule encoding the modified reporter protein or fusion protein of theinvention, and a vector capable of expressing the nucleic acid moleculeof the invention in a host cell. Preferably, the expression cassettecomprises a promoter, e.g., a constitutive or regulatable promoter,operably linked to the nucleic acid sequence. In one embodiment, theexpression cassette contains an inducible promoter. Also provided is ahost cell, e.g., a prokaryotic cell or an eukaryotic cell such as aplant or vertebrate cell, e.g., a mammalian cell, including but notlimited to a human, non-human primate, canine, feline, bovine, equine,ovine or rodent (e.g., rabbit, rat, ferret or mouse) cell, whichcomprises the expression cassette or vector of the invention, and a kitwhich comprises the nucleic acid molecule, expression cassette, vector,host cell or modified beetle luciferase or fusion protein of theinvention.

A modified reporter protein of the invention may be employed inapplications where unmodified reporter proteins cannot, such as, as afunctional reporter to measure or detect various conditions and/ormolecules of interest. For instance, a vector encoding a modified beetleluciferase comprising an insertion of a protease cleavage recognitionsite, or the modified beetle luciferase, is introduced to a cell, celllysate, in vitro transcription/translation mixture, or supernatant, andthe activity of the modified beetle luciferase detected or determined,e.g., at one or more time points and relative to a correspondingunmodified beetle luciferase. An alteration in luminescent activity inthe cell, cell lysate, in vitro transcription/translation mixture, orsupernatant over time, and/or relative to a control, e.g., a cell havingthe corresponding unmodified beetle luciferase, indicates the presenceof the protease. For instance, the invention includes a method to detecta virus associated with severe acute respiratory syndrome. The methodincludes contacting a biological, e.g., a physiological tissue or fluid,sample with a modified reporter protein, e.g., a modified beetleluciferase, comprising an internal insertion relative to a correspondingunmodified reporter protein, which modified reporter protein has adetectable activity. The insertion is at a residue or in a region in thereporter protein sequence which is tolerant to modification andcomprises an amino acid recognition sequence for a protease of thevirus. It is detected or determined whether the activity of the modifiedreporter protein in the presence of the sample is altered, therebyindicating whether the sample contains the virus.

The invention also provides a method of detecting the presence of amolecule of interest. For instance, a cell is contacted with a vectorcomprising a promoter, e.g., a regulatable promoter, and a nucleic acidsequence encoding a modified reporter protein of the invention whichcomprises an insertion which interacts with the molecule of interest. Inone embodiment, a transfected cell is cultured under conditions in whichthe promoter induces transient expression of the modified reporterprotein, and a detectable activity at the modified reporter proteindetermined.

Also provided is a method to prepare a selected mutated polynucleotideencoding a modified reporter protein. The method includes mutating aparent polynucleotide encoding a modified reporter protein withdetectable activity to yield one or more mutated polynucleotidesencoding a mutated modified reporter protein. The parent polynucleotidecomprises an open reading frame for the modified reporter protein whichis modified relative to a corresponding unmodified reporter protein at aresidue or in a region which is tolerant to modification. The modifiedreporter protein comprises an amino acid sequence which directly orindirectly interacts with a molecule of interest or is otherwisesensitive to conditions relative to the corresponding unmodifiedreporter protein. One or more mutated polynucleotides are selected whichencode mutated modified reporter proteins that have an alteredinteraction with the molecule of interest or altered activity undercertain conditions relative to the interaction or activity of themodified reporter protein. In another embodiment, the invention providesa method which includes contacting a modified reporter protein of theinvention with a library of molecules, and detecting or determiningwhether one or more molecules interacts with the modification or anon-reporter protein sequence in the modified reporter protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Overview of the EZ::TN in frame linker insertion protocol.

FIG. 2. Results for Tn5 insertion mutagenesis into the cbg69 gene. Theprotein encoded by cbg69 has one amino acid substitution at position 409(I409V) relative to a wild-type click beetle luciferase (see FIG. 3).

FIG. 3. Positions of Tn5 insertions (bolded) in a click beetleluciferase (SEQ ID NO:89).

FIG. 4. Activity of click beetle luciferases modified with a Tn5insertion.

FIGS. 5A-C. Activity of a click beetle luciferase modified with acaspase-3 recognition site insertion (cbg69DEVD). A) Relative lightunits (RLU) in a caspase assay with cbg69ss or cbg69DEVD. B) RLU in acaspase assay with click beetle luciferases and a caspase inhibitor(Ac-DEVD-CHO). C) RLU over time in an assay with varying amounts ofcaspase-3 and cbg69DEVD.

FIG. 6A. Sequence and activity of click beetle luciferases withmodifications in the hinge region, including a protease recognitionsite, a kinase recognition site, an antibody binding site, and a metalbinding site. 6 HIS=6×His-tag; FLAG=DYKDDDDK (SEQ ID NO:4); DEVD (SEQ IDNO:106)=site recognized by caspases 3/7, Pka=Pka kinase site (SEQ IDNOs. 90-96). Insertions were introduced in the hinge region of CbgLuc(1409V) using SnaBI and SalI.

FIG. 6B. SARS virus 3CL protease activity in the presence of modifiedclick beetle luciferases having SARS virus protease recognition sites.

FIG. 6C. Sequence and activity of click beetle luciferases with SARSvirus protease recognition sites in the hinge region (SEQ ID Nos. 90-91and 97-102). Insertions were introduced in the hinge region of CbgLuc(1409V) using SnaBI and SalI.

FIGS. 7A-D. Activity of firefly luciferases modified with anenterokinase recognition site. A) Amino acid sequence of a parental(unmodified) firefly luciferase (luc+) (SEQ ID NO: 103). B) RLU in anenterokinase assay with a modified firefly luciferase having aGly(3)Asp(4)LysGly(3) insertion after residue 233 or the parentalfirefly luciferase (WT). C) RLU in an enterokinase assay with a modifiedfirefly luciferase having a ProGlyProGly(3)Asp(4)LysGly(3)ProGlyProinsertion after residue 233 or the parental firefly luciferase (WT). D)RLU in an enterokinase assay with a modified firefly luciferase havingan insertion Asp(4)Lys after residue 541 or the parental fireflyluciferase (WT).

FIG. 8. Enterokinase activation of a circularly permuted fireflyluciferase having an enterokinase site.

FIG. 9. Caspase-3 activation over time by a circularly permuted fireflyluciferase having a caspase-3 site.

FIG. 10A. RLU in a caspase assay with various amounts of caspase-3 and acircularly permuted firefly luciferase having a caspase-3 recognitionsite.

FIG. 10B. RLU in a caspase assay with various amounts of caspase-3 and acircularly permuted firefly luciferase having a caspase-3 recognitionsite.

FIG. 11. Comparison of data for a circularly permuted firefly luciferasehaving an enterokinase site or a caspase-3 site.

FIG. 12. Graphs showing SARS virus 3CL protease activity with circularlypermuted click beetle (CP1: R=Asn401 and CP2: R=Arg223) and firefly (CP:R=Asp234) luciferases having SARS virus protease recognition sites.

FIG. 13. RLU for a circularly permuted luciferase having a caspase-3site, which was treated with TRAIL.

FIG. 14. Schematic of vectors for a dual luciferase caspase assay.

FIG. 15. Schematic of pBIND vector and control luciferase construct andN- or C-terminal luciferase constructs for self assembly.

FIG. 16A. SDS-PAGE analysis of full-length firefly luciferase,N-terminal portion of firefly luciferase, C-terminal portion of fireflyluciferase or a mixture of the N-terminal and C-terminal portions.

FIG. 16B. In vitro activity of full-length firefly luciferase,N-terminal portion of firefly luciferase, C-terminal portion of fireflyluciferase or a mixture of the N-terminal and C-terminal portions.

FIG. 17. In vivo activity of luciferase proteins in CHO or 293 mammaliancell extracts.

FIG. 18. Cloning strategy for preparing constructs to express fusions ofluciferase with binding partners X or Y.

FIG. 19. SDS-PAGE gel analysis of unmodified luciferase protein andfusions of luciferase with one or more heterologous sequences generatedusing an in vitro transcription/translation reaction.

FIG. 20A. Luciferase activity of unmodified luciferase (Luc2),luciferase fused to FRB (rapamycin binding protein), luciferase fused toFKBP (FK506 binding protein) and luciferase fused to FRB and FKBP, inthe presence or absence of rapamycin.

FIG. 20B. Luciferase activity of a fusion of luciferase and FRB and FKBPin the presence of increasing concentrations of FK506.

FIG. 21A. SDS-PAGE analysis of fusions of firefly luciferase (Luc2),click beetle luciferase (Cbg and Cbr) and Renilla (RLuc) luciferase withFRB and FKBP.

FIG. 21B. Luciferase activity of FRB and FKBP fusions with fireflyluciferase, click beetle luciferases and Renilla luciferase, in thepresence or absence of rapamycin.

FIG. 22. Construct for expressing luciferase from a TK promoter.

FIG. 23. Titration of FK506 in the presence of rapamycin in D293 cellstransfected with luciferase fused to FRB and FKBP (FRB1-luc2-FKBP),demonstrating inhibition of rapamycin-mediated modulation by FK506.

FIG. 24. Relative luminescence over time in D293 cells transfected witha construct with a TK promoter and a coding region for aFRB-luciferase-FKBP fusion, in the presence or absence of rapamycin.

FIG. 25A-D. Relative luminescence over time in D293 cells transfectedwith a construct with a CMV promoter linked to a coding region for aFRB-luciferase-FKBP fusion (A), a FRB-luciferase fusion (B), luciferase(C), or a luciferase-FKBP fusion (D), in the presence or absence ofrapamycin.

FIG. 26. Relative luminescence of a calmodulin-luciferase fusion in thepresence of EGTA or Ca²⁺.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “nucleic acid molecule”, “polynucleotide”, or “nucleic acidsequence” as used herein, refers to nucleic acid, DNA or RNA, thatcomprises coding sequences necessary for the production of a polypeptideor protein precursor. The encoded polypeptide may be a full-lengthpolypeptide, a fragment thereof (less than full-length), or a fusion ofeither the full-length polypeptide or fragment thereof with anotherpolypeptide, yielding a fusion polypeptide.

A “nucleic acid”, as used herein, is a covalently linked sequence ofnucleotides in which the 3′ position of the pentose of one nucleotide isjoined by a phosphodiester group to the 5′ position of the pentose ofthe next, and in which the nucleotide residues (bases) are linked inspecific sequence, i.e., a linear order of nucleotides. A“polynucleotide”, as used herein, is a nucleic acid containing asequence that is greater than about 100 nucleotides in length. An“oligonucleotide” or “primer”, as used herein, is a short polynucleotideor a portion of a polynucleotide. An oligonucleotide typically containsa sequence of about two to about one hundred bases. The word “oligo” issometimes used in place of the word “oligonucleotide”.

Nucleic acid molecules are said to have a “5′-terminus” (5′ end) and a“3′-terminus” (3′ end) because nucleic acid phosphodiester linkagesoccur to the 5′ carbon and 3′ carbon of the pentose ring of thesubstituent mononucleotides. The end of a polynucleotide at which a newlinkage would be to a 5′ carbon is its 5′ terminal nucleotide. The endof a polynucleotide at which a new linkage would be to a 3′ carbon isits 3′ terminal nucleotide. A terminal nucleotide, as used herein, isthe nucleotide at the end position of the 3′- or 5′-terminus.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotides referred to as the “5′end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring.

As used herein, a nucleic acid sequence, even if internal to a largeroligonucleotide or polynucleotide, also may be said to have 5′ and 3′ends. In either a linear or circular DNA molecule, discrete elements arereferred to as being “upstream” or 5′ of the “downstream” or 3′elements. This terminology reflects the fact that transcription proceedsin a 5′ to 3′ fashion along the DNA strand. Typically, promoter andenhancer elements that direct transcription of a linked gene (e.g., openreading frame or coding region) are generally located 5′ or upstream ofthe coding region. However, enhancer elements can exert their effecteven when located 3′ of the promoter element and the coding region.Transcription termination and polyadenylation signals are located 3′ ordownstream of the coding region.

The term “codon” as used herein, is a basic genetic coding unit,consisting of a sequence of three nucleotides that specify a particularamino acid to be incorporated into a polypeptide chain, or a start orstop signal. The term “coding region” when used in reference tostructural gene refers to the nucleotide sequences that encode the aminoacids found in the nascent polypeptide as a result of translation of amRNA molecule. Typically, the coding region is bounded on the 5′ side bythe nucleotide triplet “ATG” which encodes the initiator methionine andon the 3′ side by a stop codon (e.g., TAA, TAG, TGA). In some cases thecoding region is also known to initiate by a nucleotide triplet “TTG”.

The term “gene” refers to a DNA sequence that comprises coding sequencesand optionally control sequences necessary for the production of apolypeptide from the DNA sequence.

As used herein, the term “heterologous” nucleic acid sequence or proteinrefers to a sequence that relative to a reference sequence has adifferent source, e.g., originates from a foreign species, or, if fromthe same species, it may be substantially modified from the originalform.

Nucleic acids are known to contain different types of mutations. A“point” mutation refers to an alteration in the sequence of a nucleotideat a single base position from the wild-type sequence. Mutations mayalso refer to insertion or deletion of one or more bases, so that thenucleic acid sequence differs from a reference, e.g., a wild-type,sequence.

As used herein, the terms “hybridize” and “hybridization” refer to theannealing of a complementary sequence to the target nucleic acid, i.e.,the ability of two polymers of nucleic acid (polynucleotides) containingcomplementary sequences to anneal through base pairing. The terms“annealed” and “hybridized” are used interchangeably throughout, and areintended to encompass any specific and reproducible interaction betweena complementary sequence and a target nucleic acid, including binding ofregions having only partial complementarity. Certain bases not commonlyfound in natural nucleic acids may be included in the nucleic acids ofthe present invention and include, for example, inosine and7-deazaguanine. Those skilled in the art of nucleic acid technology candetermine duplex stability empirically considering a number of variablesincluding, for example, the length of the complementary sequence, basecomposition and sequence of the oligonucleotide, ionic strength andincidence of mismatched base pairs. The stability of a nucleic acidduplex is measured by the melting temperature, or “T_(m)”. The T_(m) ofa particular nucleic acid duplex under specified conditions is thetemperature at which on average half of the base pairs havedisassociated.

The term “recombinant DNA molecule” means a hybrid DNA sequencecomprising at least two nucleotide sequences not normally found togetherin nature.

The term “vector” is used in reference to nucleic acid molecules intowhich fragments of DNA may be inserted or cloned and can be used totransfer DNA segment(s) into a cell and capable of replication in acell. Vectors may be derived from plasmids, bacteriophages, viruses,cosmids, and the like.

The terms “recombinant vector” and “expression vector” as used hereinrefer to DNA or RNA sequences containing a desired coding sequence andappropriate DNA or RNA sequences necessary for the expression of theoperably linked coding sequence in a particular host organism.Prokaryotic expression vectors include a promoter, a ribosome bindingsite, an origin of replication for autonomous replication in a host celland possibly other sequences, e.g. an optional operator sequence,optional restriction enzyme sites. A promoter is defined as a DNAsequence that directs RNA polymerase to bind to DNA and to initiate RNAsynthesis. Eukaryotic expression vectors include a promoter, optionallya polyadenlyation signal and optionally an enhancer sequence.

A polynucleotide having a nucleotide sequence encoding a protein orpolypeptide means a nucleic acid sequence comprising the coding regionof a gene, or in other words the nucleic acid sequence encodes a geneproduct. The coding region may be present in either a cDNA, genomic DNAor RNA form. When present in a DNA form, the oligonucleotide may besingle-stranded (i.e., the sense strand) or double-stranded. Suitablecontrol elements such as enhancers/promoters, splice junctions,polyadenylation signals, etc. may be placed in close proximity to thecoding region of the gene if needed to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in the expression vectors ofthe present invention may contain endogenous enhancers/promoters, splicejunctions, intervening sequences, polyadenylation signals, etc. Infurther embodiments, the coding region may contain a combination of bothendogenous and exogenous control elements.

The term “transcription regulatory element” or “transcription regulatorysequence” refers to a genetic element or sequence that controls someaspect of the expression of nucleic acid sequence(s). For example, apromoter is a regulatory element that facilitates the initiation oftranscription of an operably linked coding region. Other regulatoryelements include, but are not limited to, transcription factor bindingsites, splicing signals, polyadenylation signals, termination signalsand enhancer elements.

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription. Promoter and enhancer elements have been isolated froma variety of eukaryotic sources including genes in yeast, insect andmammalian cells. Promoter and enhancer elements have also been isolatedfrom viruses and analogous control elements, such as promoters, are alsofound in prokaryotes. The selection of a particular promoter andenhancer depends on the cell type used to express the protein ofinterest. Some eukaryotic promoters and enhancers have a broad hostrange while others are functional in a limited subset of cell types. Forexample, the SV40 early gene enhancer is very active in a wide varietyof cell types from many mammalian species and has been widely used forthe expression of proteins in mammalian cells. Two other examples ofpromoter/enhancer elements active in a broad range of mammalian celltypes are those from the human elongation factor 1 gene and the longterminal repeats of the Rous sarcoma virus; and the humancytomegalovirus.

The term “promoter/enhancer” denotes a segment of DNA containingsequences capable of providing both promoter and enhancer functions(i.e., the functions provided by a promoter element and an enhancerelement as described above). For example, the long terminal repeats ofretroviruses contain both promoter and enhancer functions. Theenhancer/promoter may be “endogenous” or “exogenous” or “heterologous.”An “endogenous” enhancer/promoter is one that is naturally linked with agiven gene in the genome. An “exogenous” or “heterologous”enhancer/promoter is one that is placed in juxtaposition to a gene bymeans of genetic manipulation (i.e., molecular biological techniques)such that transcription of the gene is directed by the linkedenhancer/promoter.

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript ineukaryotic host cells. Splicing signals mediate the removal of intronsfrom the primary RNA transcript and consist of a splice donor andacceptor site. A commonly used splice donor and acceptor site is thesplice junction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly(A) site” or“poly(A) sequence” as used herein denotes a DNA sequence which directsboth the termination and polyadenylation of the nascent RNA transcript.Efficient polyadenylation of the recombinant transcript is desirable, astranscripts lacking a poly(A) tail are unstable and are rapidlydegraded. The poly(A) signal utilized in an expression vector may be“heterologous” or “endogenous.” An endogenous poly(A) signal is one thatis found naturally at the 3′ end of the coding region of a given gene inthe genome. A heterologous poly(A) signal is one which has been isolatedfrom one gene and positioned 3′ to another gene. A commonly usedheterologous poly(A) signal is the SV40 poly(A) signal. The SV40 poly(A)signal is contained on a 237 bp BamH I/Bcl I restriction fragment anddirects both termination and polyadenylation.

Eukaryotic expression vectors may also contain “viral replicons” or“viral origins of replication.” Viral replicons are viral DNA sequencesthat allow for the extrachromosomal replication of a vector in a hostcell expressing the appropriate replication factors. Vectors containingeither the SV40 or polyoma virus origin of replication replicate to highcopy number (up to 10⁴ copies/cell) in cells that express theappropriate viral T antigen. In contrast, vectors containing thereplicons from bovine papillomavirus or Epstein-Barr virus replicateextrachromosomally at low copy number (about 100 copies/cell).

The term “in vitro” refers to an artificial environment and to processesor reactions that occur within an artificial environment. In vitroenvironments include, but are not limited to, test tubes and celllysates. The term “in vivo” refers to the natural environment (e.g., ananimal or a cell) and to processes or reaction that occur within anatural environment.

The term “expression system” refers to any assay or system fordetermining (e.g., detecting) the expression of a gene of interest.Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems may be used. A wide range ofsuitable mammalian cells are available from a wide range of source(e.g., the American Type Culture Collection, Rockland, Md.). The methodof transformation or transfection and the choice of expression vehiclewill depend on the host system selected. Transformation and transfectionmethods are well known to the art. Expression systems include in vitrogene expression assays where a gene of interest (e.g., a reporter gene)is linked to a regulatory sequence and the expression of the gene ismonitored following treatment with an agent that inhibits or inducesexpression of the gene. Detection of gene expression can be through anysuitable means including, but not limited to, detection of expressedmRNA or protein (e.g., a detectable product of a reporter gene) orthrough a detectable change in the phenotype of a cell expressing thegene of interest. Expression systems may also comprise assays where acleavage event or other nucleic acid or cellular change is detected.

The term “wild-type” as used herein, refers to a gene or gene productthat has the characteristics of that gene or gene product isolated froma naturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “wild-type” form of the gene. In contrast, the term “mutant” refersto a gene or gene product that displays modifications in sequence and/orfunctional properties (i.e., altered characteristics) when compared tothe wild-type gene or gene product. It is noted that naturally-occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics when compared to the wild-type gene or geneproduct.

The term “isolated” when used in relation to a nucleic acid, as in“isolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant with which it is ordinarily associated in its source. Thus,an isolated nucleic acid is present in a form or setting that isdifferent from that in which it is found in nature. In contrast,non-isolated nucleic acids (e.g., DNA and RNA) are found in the statethey exist in nature. For example, a given DNA sequence (e.g., a gene)is found on the host cell chromosome in proximity to neighboring genes;RNA sequences (e.g., a specific mRNA sequence encoding a specificprotein), are found in the cell as a mixture with numerous other mRNAsthat encode a multitude of proteins. However, isolated nucleic acidincludes, by way of example, such nucleic acid in cells ordinarilyexpressing that nucleic acid where the nucleic acid is in a chromosomallocation different from that of natural cells, or is otherwise flankedby a different nucleic acid sequence than that found in nature. Theisolated nucleic acid or oligonucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acidor oligonucleotide is to be utilized to express a protein, theoligonucleotide contains at a minimum, the sense or coding strand (i.e.,the oligonucleotide may single-stranded), but may contain both the senseand anti-sense strands (i.e., the oligonucleotide may bedouble-stranded).

By “peptide,” “protein” and “polypeptide” is meant any chain of aminoacids, regardless of length or post-translational modification (e.g.,glycosylation or phosphorylation). The nucleic acid molecules of theinvention may also encode a variant of a naturally-occurring protein orpolypeptide fragment thereof, which has an amino acid sequence that isat least 85%, 90%, 95% or 99% identical to the amino acid sequence ofthe naturally-occurring (native or wild-type) protein from which it isderived. The term “fusion polypeptide” or “fusion protein” refers to achimeric protein containing a reference protein (e.g., luciferase)joined at the N- and/or C-terminus to one or more heterologous sequences(e.g., a non-luciferase polypeptide). In some embodiments, a modifiedpolypeptide, fusion polypeptide or a portion of a full-lengthpolypeptide of the invention, may retain at least some of the activityof a corresponding full-length functional (nonchimeric) polypeptide. Inother embodiments, in the absence of an exogenous agent or molecule ofinterest, a modified polypeptide, fusion polypeptide or portion of afull-length functional polypeptide of the invention, may lack activityrelative to a corresponding full-length functional polypeptide. In otherembodiments, a modified polypeptide, fusion polypeptide or portion of afull-length functional polypeptide of the invention in the presence ofan exogenous agent may retain at least some or have substantially thesame activity, or alternatively lack activity, relative to acorresponding full-length functional polypeptide.

Polypeptide molecules are said to have an “amino terminus” (N-terminus)and a “carboxy terminus” (C-terminus) because peptide linkages occurbetween the backbone amino group of a first amino acid residue and thebackbone carboxyl group of a second amino acid residue. The terms“N-terminal” and “C-terminal” in reference to polypeptide sequencesrefer to regions of polypeptides including portions of the N-terminaland C-terminal regions of the polypeptide, respectively. A sequence thatincludes a portion of the N-terminal region of polypeptide includesamino acids predominantly from the N-terminal half of the polypeptidechain, but is not limited to such sequences. For example, an N-terminalsequence may include an interior portion of the polypeptide sequenceincluding bases from both the N-terminal and C-terminal halves of thepolypeptide. The same applies to C-terminal regions. N-terminal andC-terminal regions may, but need not, include the amino acid definingthe ultimate N-terminus and C-terminus of the polypeptide, respectively.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule expressed from a recombinant DNAmolecule. In contrast, the term “native protein” is used herein toindicate a protein isolated from a naturally occurring (i.e., anonrecombinant) source. Molecular biological techniques may be used toproduce a recombinant form of a protein with identical properties ascompared to the native form of the protein.

The terms “cell,” “cell line,” “host cell,” as used herein, are usedinterchangeably, and all such designations include progeny or potentialprogeny of these designations. By “transformed cell” is meant a cellinto which (or into an ancestor of which) has been introduced a nucleicacid molecule of the invention. Optionally, a nucleic acid molecule ofthe invention may be introduced into a suitable cell line so as tocreate a stably-transfected cell line capable of producing the proteinor polypeptide encoded by the gene. Vectors, cells, and methods forconstructing such cell lines are well known in the art. The words“transformants” or “transformed cells” include the primary transformedcells derived from the originally transformed cell without regard to thenumber of transfers. All progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Nonetheless, mutantprogeny that have the same functionality as screened for in theoriginally transformed cell are included in the definition oftransformants.

The term “homology” refers to a degree of complementarity between two ormore sequences. There may be partial homology or complete homology(i.e., identity). Homology is often measured using sequence analysissoftware (e.g., Sequence Analysis Software Package of the GeneticsComputer Group. University of Wisconsin Biotechnology Center. 1710University Avenue. Madison, Wis. 53705). Such software matches similarsequences by assigning degrees of homology to various substitutions,deletions, insertions, and other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid, asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine.

The term “isolated” when used in relation to a polypeptide, as in“isolated protein” or “isolated polypeptide” refers to a polypeptidethat is identified and separated from at least one contaminant withwhich it is ordinarily associated in its source. Thus, an isolatedpolypeptide is present in a form or setting that is different from thatin which it is found in nature. In contrast, non-isolated polypeptides(e.g., proteins and enzymes) are found in the state they exist innature.

The term “purified” or “to purify” means the result of any process thatremoves some of a contaminant from the component of interest, such as aprotein or nucleic acid. The percent of a purified component is therebyincreased in the sample.

As used herein, “pure” means an object species is the predominantspecies present (i.e., on a molar basis it is more abundant than anyother individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a “substantially pure”composition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, about 90%, about 95%, and about 99%. Most preferably,the object species is purified to essential homogeneity (contaminantspecies cannot be detected in the composition by conventional detectionmethods) wherein the composition consists essentially of a singlemacromolecular species.

The term “operably linked” as used herein refer to the linkage ofnucleic acid sequences in such a manner that a nucleic acid moleculecapable of directing the transcription of a given gene and/or thesynthesis of a desired protein molecule is produced. The term alsorefers to the linkage of sequences encoding amino acids in such a mannerthat a functional (e.g., enzymatically active, capable of binding to abinding partner, capable of inhibiting, etc.) protein or polypeptide isproduced.

As used herein, the term “poly-histidine tract” or (His tag) refers to amolecule comprising two to ten histidine residues, e.g., apoly-histidine tract of five to ten residues. A poly-histidine tractallows the affinity purification of a covalently linked molecule on animmobilized metal, e.g., nickel, zinc, cobalt or copper, chelate columnor through an interaction with another molecule (e.g., an antibodyreactive with the His tag).

A “protein destabilization sequence” includes, but is not limited to, aPEST sequence, for example, a PEST sequence from cyclin, e.g., mitoticcyclins, uracil permease or ODC, a sequence from the C-terminal regionof a short-lived protein such as ODC, early response proteins such ascytokines, lymphokines, protooncogenes, e.g., c-myc or c-fos, MyoD, HMGCoA reductase, or S-adenosyl methionine decarboxylase, CL sequences, acyclin destruction box, or N-degron.

As used herein, a “marker gene” or “reporter gene” is a gene thatimparts a distinct phenotype to cells expressing the gene and thuspermits cells having the gene to be distinguished from cells that do nothave the gene. Such genes may encode either a selectable or screenablemarker, depending on whether the marker confers a trait which one can‘select’ for by chemical means, i.e., through the use of a selectiveagent (e.g., a herbicide, antibiotic, or the like), or whether it issimply a “reporter” trait that one can identify through observation ortesting, i.e., by ‘screening’. Elements of the present disclosure areexemplified in detail through the use of particular marker genes. Ofcourse, many examples of suitable marker genes or reporter genes areknown to the art and can be employed in the practice of the invention.Therefore, it will be understood that the following discussion isexemplary rather than exhaustive. In light of the techniques disclosedherein and the general recombinant techniques which are known in theart, the present invention renders possible the alteration of any gene.Exemplary modified reporter proteins are encoded by nucleic acidmolecules comprising modified reporter genes including, but are notlimited to, modifications of a neo gene, a β-gal gene, a gus gene, a catgene, a gpt gene, a hyg gene, a hisD gene, a ble gene, a mprt gene, abar gene, a nitrilase gene, a galactopyranoside gene, a xylosidase gene,a thymidine kinase gene, an arabinosidase gene, a mutant acetolactatesynthase gene (ALS) or acetoacid synthase gene (AAS), amethotrexate-resistant dhfr gene, a dalapon dehalogenase gene, a mutatedanthranilate synthase gene that confers resistance to 5-methyltryptophan (WO 97/26366), an R-locus gene, a β-lactamase gene, a xylEgene, an α-amylase gene, a tyrosinase gene, a luciferase (luc) gene,(e.g., a Renilla reniformis luciferase gene, a firefly luciferase gene,or a click beetle luciferase (Pyrophorus plagiophthalamus) gene), anaequorin gene, a red fluorescent protein gene, or a green fluorescentprotein gene.

All amino acid residues identified herein are in the naturalL-configuration. In keeping with standard polypeptide nomenclature,abbreviations for amino acid residues are as shown in the followingTable of Correspondence.

TABLE OF CORRESPONDENCE 1-Letter 3-Letter AMINO ACID Y Tyr L-tyrosine GGly L-glycine F Phe L-phenylalanine M Met L-methionine A Ala L-alanine SSer L-serine I Ile L-isoleucine L Leu L-leucine T Thr L-threonine V ValL-valine P Pro L-proline K Lys L-lysine H His L-histidine Q GlnL-glutamine E Glu L-glutamic acid W Trp L-tryptophan R Arg L-arginine DAsp L-aspartic acid N Asn L-asparagine C Cys L-cysteineI. Methods to Identify Residues or Regions of a Reporter Protein whichare Tolerant to Modification

Numerous methods are available to identify sites and/or regions in areporter protein gene which may be modified, e.g., disrupted, yet whentranscribed and translated, yield a desirable, for instance, a readilydetectable, gene product. For instance, amplification reactions may beemployed to delete and/or insert nucleotides for one or more amino acidresidues in a reporter protein gene. Alternatively, transposons may beemployed to prepare libraries of insertional mutations. Transposons aremobile DNA sequences found in the genomes of prokaryotes and eukaryotes.Transposon tagging has long been recognized as a powerful research toolfor randomly distributing primer binding sites, creating gene“knockouts,” and introducing a physical tag or a genetic tag into largetarget DNAs. Insertions in a reporter gene useful to prepare themodified reporter proteins of the invention are those which areinternal, in frame insertions in the coding region for the reporterprotein. The following examples, which are for illustration only,describe the use of a Tn5-based system (EZ::TN™ from Epicentre, Madison,Wis.) and a Tn7-based system (GPS-M Mutagenesis System, New EnglandBiolabs, Inc.) to identify regions in a reporter gene which are tolerantto insertions.

A. Tn-5 Insertional Mutagenesis

One frequently used transposition system is the Tn5 system isolated fromgram-negative bacteria. The Tn5 transposase is a small, single subunitenzyme that has been cloned and purified to high specific activity, andcarries out transposition without the need for host cell factors.Moreover, Tn5 transposon insertions into target DNA are highly random,and proceed by a simple process. Tn5 transposase will transpose any DNAsequence contained between its short 19 basepair Mosaic End (ME) Tn5transposase recognition sequences. An overview of the EZ::TN in framelinker insertion protocol is shown in FIG. 1.

i. Transposon Insertion Reaction

Target DNA Preparation

The target reporter DNA is selected as one which is not encoded by atransposon gene, e.g., a kanamycin resistance gene. While the transposoninsertion reaction is not significantly inhibited by high levels of RNAcontamination in target DNA preparations, if the target DNA is heavilycontaminated with chromosomal DNA, which is a direct competitor fortarget transposition, the number of clones is reduced. Plasmid andcosmid clones can be purified by standard minilysate procedures and usedas target DNA in the insertion reaction. Low copy-number vectors, forexample, BAC or cosmid clones, are often contaminated with a highermolar proportion of E. coli chromosomal DNA, thus reducing thetransposon insertion frequency. Therefore, it is preferred BAC andcosmid DNA are purified, to remove the chromosomal DNA prior to theinsertion reaction.

In Vitro Transposon Insertion Reaction

Reaction conditions are optimized to maximize the efficiency of thetransposon insertion while minimizing multiple insertion events. Forexample, an equimolar amount of the transposon is added to the moles oftarget DNA.

-   -   1. Prepare the transposon insertion reaction mixture by adding        in the following order:        -   1 μl 10× reaction buffer        -   0.2 μg target DNA*        -   x μl molar equivalent transposon        -   x μl sterile water to a reaction volume of 9 μl        -   1 μl transposase        -   10 μl total reaction volume    -   2. Incubate the reaction mixture for 2 hours at 37° C.    -   3. Stop the reaction by adding μl stop solution.    -   Mix and heat for 10 minutes at 70° C.    -   The reaction mixture may be stored at −20° C.        ii. Selection of Transposon Insertion Clones

Transformation and Recovery

The number of transposon insertion clones obtained per reaction dependson, among other factors, the transformation efficiency of the competentcells used. The greater the transformation efficiency of the competentcells, the greater the number of insertion clones obtained. A recA⁻strain of E. coli is preferred to eliminate the possibility ofgenerating multimeric forms of the vector. Also, the host strain mustnot express any antibiotic resistance marker, e.g., a kanamycinresistance marker, present in the transposon.

-   -   1. Using 1 μl of the insertion reaction mixture, transform        recA⁻ E. coli, e.g., electrocompetent cells.    -   2. Recover the electroporated cells by adding SOC medium to the        electroporation cuvette to 1 ml final volume immediately after        electroporation. Pipette the medium/cells gently to mix.        Transfer to a tube and incubate on a 37° C. shaker for 30-60        minutes to facilitate cell outgrowth.

Plating and Selecting Transformants

Transposon insertion clones are selected on antibiotic-containingplates. For Tn5, kanamycin-containing plates may be used, however, thetransposon can also confer resistance to neomycin and G418 in E. coli.

-   -   1. Plate portions of cells onto LB plates containing 50 μg/ml        kanamycin.    -   2. To determine the transposon insertion efficiency, plate        identical dilutions and dilution aliquots of the transformation        reaction on a second plate containing an antibiotic specific for        selecting target DNA (e.g., 100 μg/ml ampicillin for the control        DNA). The transposition frequency is given by the ratio of        Kan^(R)/Amp^(R) clones for the control DNA.    -   3. Grow plates overnight at 37° C. Assuming a transposon        insertion efficiency of 1% and use of high purity target DNA        (i.e., little or no chromosomal DNA contamination), there are        about 100-500 Kan^(R) clones per plate.        iii. Generating an in Frame 19 Codon Insertion

Transposon Insertion Mapping

Tn5 randomly inserts into target DNA. Therefore, the transposoninsertion site in each clone should be determined prior to restrictionendonuclease digestion, e.g., NotI digestion, by one of three methods:

1. Insertion clones can be sequenced bidirectionally using forward andreverse transposon-specific primers. The insertion site of each clonecan also be mapped prior to sequencing.

2. Insertion sites can be mapped by size analysis of PCR products usingcolony minilysate DNA as a template. To map the insertion sites, forwardor reverse transposon-specific primers and a vector-specific flankingprimers may be employed.

3. Alternatively, insertion sites can be mapped by restrictionendonuclease digest(s).

Once the transposon insertion site of the desired clones is determined,the clones are individually digested with a restriction enzyme, e.g.,NotI, to linearize the DNA. The linearized DNA is then purified (e.g.,by agarose gel electrophoresis, column purification, and the like).

Religation and Transformation

The linearized clones are religated using T4 DNA ligase. Successfulreligation regenerates a single restriction site, e.g., NotI, andcreates the 57 nucleotide (19 codon) insertion into all three readingframes. The religated DNA is transformed into selected cells andrecombinants selected using an antibiotic marker present on the originalcloning vector (e.g., ampicillin for the control DNA).

Analysis of the 19 Codon Insertion Clones

Nine of the 57 nucleotides are the result of a 9 bp sequence duplicationimmediately flanking the transposon insertion site. The amino acidsequence of the protein encoded by the target DNA is conserved on bothsides of the 19 codon insertion.

iv. DNA Sequencing of Transposon Insertion Clones

Primer Consideration

Primers should be constructed to minimize homology to commonly usedcloning vectors, and the sequence of each primer should be compared tothat of the user's specific cloning vector to ensure minimal sequencehomology to the vector.

Target Site Duplication

Tn5-catalyzed transposon insertion results in the generation of a 9 bptarget site sequence duplication where one copy immediately flanks eachside of the inserted transposon.

Distinguishing Transposon Sequence for Insert Sequence

If the primers anneal to a region near the ends of the transposon, thefirst sequence data obtained from each sequencing reaction is that ofTransposon DNA.

B. Tn7-Based Insertional Mutagenesis

The GPS-M Mutagenesis System uses TnsABC*Transposase to insert aTn7-based transposon randomly into a DNA target. Target DNA may be aplasmid, cosmid, BAC or purified chromosomal DNA. If the insertion siteis within a translated gene segment, this will normally result in a null(loss of function) mutation. There is minimal site preference forinsertion, so disruption of any open reading frame is possible. Due totarget immunity, only one insertion occurs per DNA molecule in vivo overa distance of about 190 kb. Therefore, the in vitro reaction produces apopulation of target DNA molecules each containing the transposableelement at a different position.

The transposon donor can be modified by adding to or replacing theantibiotic, e.g., kanamycin, resistance marker. The donor plasmid may begrown in standard laboratory E. coli strains, and the vector backbonecarries a different antibiotic marker, e.g., Amp^(r), than thetransposon and an origin of replication. To destroy unreacted donormolecules and avoid undesirable reaction products, the donor can bedestroyed by digestion with a rare-cutting enzyme, for instance, PI-SceI(VDE). For applications in which the mutagenized DNA is transformed intonaturally-competent organisms (which take up single DNA strands), thegaps are filled-in and ligated.

i. Reaction Protocol

-   -   1. Mix the following reagents (per 20 μl reaction):

2 μl 10 X buffer 1 μl supercoiled custom donor (0.02 μg) 0.08 μg targetDNA dH₂O 18 μl Total Volume

Mix well by pipetting up and down a few times.

-   -   2. Add 1 μl transposase to each tube. Mix again.    -   3. Incubate for 10 minutes at 37° C. This is the assembly        reaction.    -   4. Add 1 μl start solution to each tube. Mix well by pipetting        up and down a few times.    -   5. Incubate for 1 hour at 37° C. This is the strand transfer        reaction.    -   6. Heat inactivate at 75° C. for 10 minutes. Note: 65° C. is not        adequate.    -   7. Optional gap repair.    -   8. Add 5 μl 10× Pl-SceI Buffer        -   0.5 μl BSA        -   18.5 μl dH₂O        -   6 μl Pl-SceI (VDE) (6 units)    -   9. Incubate for 1-2 hours at 37° C.    -   10. Incubate for 10 minutes at 75° C.    -   11. Transform. For chemical transformation with subcloning        efficiency cells (10⁷ per microgram of pUC), transform 1 μl and        10 μl of undiluted reaction. For electroporation (>10⁹ per        microgram of pUC), dilute 10-fold in dH₂O and transform 1 μl and        10 μl. To outgrow, dilute the transformation mixture into 1 ml        LB or as directed by the manufacturer, and incubate for 1 hour        at 37° C. with aeration. This period without selection is        necessary for expression of drug resistance, especially        kanamycin.        ii. General Considerations

Amount of Target

The recommended mass of target DNA (0.08 μg per reaction) works well forplasmid targets. For cosmids and BACs, a molar ratio of around 2:1(donor to target) works well. Increasing the ratio to 4:1 decreases theefficiency slightly.

Donor:Target Ratio

The recommended donor:target mass ratio (1:4, 0.08 μg target per 20 μlreaction) is optimal. Small deviations produce only small changes in thenumber of recovered products. However, saturating amounts of donorinhibit the reaction and may lead to accumulation of double insertions.

Order of Addition

Water, target DNA, buffer and donor plasmid should be added first,followed by transposase. The start solution should be added only afterthe assembly reaction.

Assembly Reaction

If this step is omitted, the proportion of complicated products isincreased.

Time of Incubation

The reaction is linear at 37° C. for at least one hour. Extremely longincubation times may lead to accumulation of double insertions.

Temperature of Incubation

The reaction proceeds, but more slowly, at room temperature and at 30°C. For reactions with BACs, 30° C. is recommended.

Heat Killing

Heating at 75° C. for 10 minutes effectively disrupts the reactioncomplexes. Heating at 65° C. for 20 minutes is not adequate.Phenol/chloroform extraction followed by alcohol precipitation is alsoeffective.

Scaling the Procedure

Increase or reduce the final volume and the volume of all components bythe same percentage; the relative concentrations of the two DNA speciesand the proteins are very important, as are the buffer conditions.

Enzyme Names

Pl-SceI (VDE) is not the same as l-SceI. Use Pl-SceI (VDE) to digest thedonor and SceI for mapping insertions obtained.

Gap Repair

This step is not required for transformation into E. coli and isnecessary only when the desired application involves transformation intonaturally competent bacteria. Naturally competent bacteria includemembers of the genera Neisseria, Haemophilus, Bacillus, Pneumococcus,Staphylococcus, and Streptococcus. DNA uptake into these organismsinvolves degradation of one strand, concomitant with internalization ofthe other strand. Without gap repair, the 5-base gaps at the transposoninsertion site will unlink the transposon insertion from flanking DNA onone side or the other. Organisms in which competence is inducedchemically or by electroporation (e.g., E. coli and other entericbacteria tissue culture cells, etc.) take up both DNA strands. Gaps atthe insertion site are efficiently repaired by the cellular machinery.

iii. GAP Repair Protocol

-   -   7. Phenol/chloroform extract (501).    -   8. Ethanol precipitate:        -   6 μl 3M NaAcetate        -   100 μl EtOH        -   Incubate for 20 minutes at −20° C.        -   Centrifuge for 10 minutes in a microfuge    -   9. Resuspend in 15 μl TE.    -   10. 1 μl DNA Polymerase I (E. coli) (10 units)        -   3 μl 10× EcoPol Buffer        -   9 μl dNTP (at 100 μM each nucleotide; final concentration 33            μM each)    -   11. Incubate for 15 minutes at room temperature.    -   12. Add 1 μl T4 DNA ligase (400 units) and ATP to a final        concentration of 1 mM.    -   13. Incubate for 4 hours at 16° C.    -   14. Phenol/chloroform extract.    -   15. Alcohol precipitate.    -   16. Resuspend in 20 μl TE.    -   17. Add 5 μl OX Pl-SceI Buffer        -   0.5 μl BSA        -   18.5 μl dH₂O        -   6 μl Pl-SceI (VDE) (6 units)    -   18. Incubate for 1-2 hours at 37° C.    -   19. Incubate for 10 minutes at 75° C.    -   20. Transform according to the appropriate method.        iv. Donor Manipulation    -   1. The transposon donor must be supercoiled. The efficiency of        reaction using a relaxed or linear donor is reduced by about        100-fold. The donor preparation should be good quality, but        CsCl-purification is not necessary.    -   2. Essential recognition elements for the transposase are not        dispensable. There may be stop codons in all frames reading into        the transposon. Transcription can proceed into the dispensable        region from outside without difficulty.    -   3. Transposition efficiency may decline somewhat as the        transposon becomes longer.    -   4. For best results, ensure that your transposon donor plasmid        is monomeric.    -   5. The Pl-SceI digestion step may be omitted if the donor        preparation is monomeric and supercoiled and if the donor        molecules will not replicate in the host organism.        v. Target DNA Requirements

Plasmid targets for sequencing should be in circular form to facilitaterecovery. Linear (e.g., chromosomal) DNA is an efficient substrate. Arepair and ligation step is required before transformation, when usingnaturally transformable organisms. Large plasmids, such as cosmids andBACs, are usable targets. Target DNA must be at least 5 μg/ml in ano-salt buffer such as 1×TE. The concentration can be estimated bycomparison of agarose gel band intensity with a DNA of knownconcentration or by absorbance at 260.

II. Exemplary Modifications

Once a site or region in a reporter protein is identified that istolerant to modification, that site or region may be modified bydeletion of one or more residues, insertion of one or more residuesand/or by circular permutation or any combination thereof. In oneembodiment, the modification may be the introduction of a recognitionsite for a hydrolase including but not limited to proteases, peptidases,esterases (e.g., cholesterol esterase), phosphatases (e.g., alkalinephosphatase) and the like. For instance, hydrolases include, but are notlimited to, enzymes acting on peptide bonds (peptide hydrolases) such asaminopeptidases, dipeptidases, dipeptidyl-peptidases andtripeptidyl-peptidases, peptidyl-dipeptidases, serine-typecarboxypeptidases, metallocarboxypeptidases, cysteine-typecarboxypeptidases, omega peptidases, serine endopeptidases, cysteineendopeptidases, aspartic endopeptidases, metalloendopeptidases,threonine endopeptidases, and endopeptidases of unknown catalyticmechanism. For example, a modified beetle luciferase of the inventionmay comprise an enterokinase cleavage site, a caspase cleavage site, acoronavirus protease site (STLQ-SGLRKMA; SEQ ID NO:10), a kinase site, aHIV-1 protease site (SQNY-PIVQ or KAVRL-AEAMS; SEQ ID NO: 11 and SEQ IDNO:12, respectively), a HCV protease site (AEDVVCC-SMSYS; SEQ ID NO:13)(see, e.g., Lee et al., 2003), a SARS virus protease site (e.g.,QTSITSAVLQSGFRKMAFPS; SEQ ID NO: 16, or VRQCSGVTFQGKFKKIVKGT; SEQ ID NO:17), a rhinovirus protease site, e.g., rhinovirus 3C protease site, aprohormone convertase site, an interleukin-16-converting enzyme site, aCMV assembling site, a leishmandysin site, B. anthracis lethal factor, abotulinum neurotoxin light chain protease site, a β-secretase site foramyloid precursor protein (VKM-DAEF; SEQ ID NO: 14), prostate specificantigen sequence, a thrombin site, a renin and angiotensin-convertingenzyme site, a cathepsin D site, a matrix metalloproteinase site, a uPAsite, a plasmin site, a binding site for a cation, such as a calciumbinding domain, a calmodulin binding domain, a cellulose binding domain,a chitin binding domain, a maltose binding protein domain, or a biotinbinding domain. In another embodiment, a modified reporter protein ofthe invention may comprise a sequence recognized by a ligand such as anantibody or a metal such as calcium.

III. Exemplary Polynucleotides and Proteins

The invention includes a modified reporter protein encompassing anyamino acid sequence which provides a polypeptide having a detectableactivity, e.g., luminescent activity, as well as protein fragmentsthereof, which are recombinantly or synthetically synthesized. Thereporter protein sequences of a modified reporter protein are the sameor are substantially the same as the amino acid sequence of acorresponding unmodified reporter protein. A polypeptide or peptidehaving substantially the same sequence means that an amino acid sequenceis largely, but may not entirely be, the same and retains a functionalactivity of the sequence to which it is related. In general, two aminoacid sequences are substantially the same or substantially homologous ifthey are at least 70% identical, e.g., have at least 80%, 90%, 95% ormore identity.

Homology or identity is often measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

Methods of alignment of sequence for comparison are well-known in theart. Optimal alignment of sequences for comparison can be conducted bythe local homology algorithm of Smith et al. (1981), by the homologyalignment algorithm of Needleman et al. (1970), by the search forsimilarity method of Person et al. (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988); Higgins et al. (1989); Corpet et al. (1988); Huang et al.(1992); and Pearson et al. (1994). The ALIGN program is based on thealgorithm of Myers and Miller (1988). The BLAST programs of Altschul etal. (1990), are based on the algorithm of Karlin and Altschul (1990).

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., 1990). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993). One measure ofsimilarity provided by the BLAST algorithm is the smallest sumprobability (P(N)), which provides an indication of the probability bywhich a match between two nucleotide or amino acid sequences would occurby chance. For example, a test nucleic acid sequence is consideredsimilar to a reference sequence if the smallest sum probability in acomparison of the test nucleic acid sequence to the reference nucleicacid sequence is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al. (1997).Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al., supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.BLASTN for nucleotide sequences, BLASTX for proteins) can be used. TheBLASTN program (for nucleotide sequences) uses as defaults a wordlength(W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989). Seehttp://www.ncbi.nlm.nih.gov.

In particular, a polypeptide may be substantially related but for aconservative variation. A conservative variation denotes the replacementof an amino acid residue by another, biologically similar residue.Examples of conservative variations include the substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, and the like. Other illustrative examplesof conservative substitutions include the changes of: alanine to serine;arginine to lysine; asparagine to glutamine or histidine; aspartate toglutamate; cysteine to serine; glutamine to asparagine; glutamate toaspartate; glycine to proline; histidine to asparagine or glutamine;isoleucine to leucine or valine; leucine to valine or isoleucine; lysineto arginine, glutamine, or glutamate; methionine to leucine orisoleucine; phenylalanine to tyrosine, leucine or methionine; serine tothreonine; threonine to serine; tryptophan to tyrosine; tyrosine totryptophan or phenylalanine; valine to isoleucine to leucine.

In one embodiment, a polynucleotide of the invention is optimized forexpression in a particular host. As used herein, optimization includescodon optimization as well as, in eukaryotic cells, introduction of aKozak sequence, and/or one or more introns. Thus, a nucleic acidmolecule may have a codon composition that differs from that of awild-type nucleic acid sequence encoding an unmodified beetle luciferaseat more than 30%, 35%, 40% or more than 45%, e.g., 50%, 55%, 60% or moreof the codons. Preferred codons for use in the invention are those whichare employed more frequently than at least one other codon for the sameamino acid in a particular organism and, more preferably, are also notlow-usage codons in that organism and are not low-usage codons in theorganism used to clone or screen for the expression of the nucleic acidmolecule. Moreover, preferred codons for certain amino acids (i.e.,those amino acids that have three or more codons,), may include two ormore codons that are employed more frequently than the other(non-preferred) codon(s). The presence of codons in the nucleic acidmolecule that are employed more frequently in one organism than inanother organism results in a nucleic acid molecule which, whenintroduced into the cells of the organism that employs those codons morefrequently, is expressed in those cells at a level that is greater thanthe expression of the wild-type or parent nucleic acid sequence in thosecells.

In one embodiment of the invention, the codons that are different arethose employed more frequently in a mammal, while in another embodimentthe codons that are different are those employed more frequently in aplant. A particular type of mammal, e.g., human, may have a differentset of preferred codons than another type of mammal. Likewise, aparticular type of plant may have a different set of preferred codonsthan another type of plant. In one embodiment of the invention, themajority of the codons which differ are ones that are preferred codonsin a desired host cell. Preferred codons for mammals (e.g., humans) andplants are known to the art (e.g., Wada et al., 1990). For example,preferred human codons include, but are not limited to, CGC (Arg), CTG(Leu), TCT (Ser), AGC (Ser), ACC (Thr), CCA (Pro), CCT (Pro), GCC (Ala),GGC (Gly), GTG (Val), ATC (Ile), ATT (Ile), AAG (Lys), AAC (Asn), CAG(Gln), CAC (His), GAG (Glu), GAC (Asp), TAC (Tyr), TGC (Cys) and TTC(Phe) (Wada et al., 1990). Thus, preferred “humanized” synthetic nucleicacid molecules of the invention have a codon composition which differsfrom a wild type nucleic acid sequence by having an increased number ofthe preferred human codons, e.g. CGC, CTG, TCT, AGC, ACC, CCA, CCT, GCC,GGC, GTG, ATC, ATT, AAG, AAC, CAG, CAC, GAG, GAC, TAC, TGC, TTC, or anycombination thereof. For example, the nucleic acid molecule of theinvention may have an increased number of CTG or TTG leucine-encodingcodons, GTG or GTC valine-encoding codons, GGC or GGT glycine-encodingcodons, ATC or ATT isoleucine-encoding codons, CCA or CCTproline-encoding codons, CGC or CGT arginine-encoding codons, AGC or TCTserine-encoding codons, ACC or ACT threonine-encoding codon, GCC or GCTalanine-encoding codons, or any combination thereof, relative to thewild-type nucleic acid sequence. Similarly, nucleic acid moleculeshaving an increased number of codons that are employed more frequentlyin plants, have a codon composition which differs from a wild-typenucleic acid sequence by having an increased number of the plant codonsincluding, but not limited to, CGC (Arg), CTT (Leu), TCT (Ser), TCC(Ser), ACC (Thr), CCA (Pro), CCT (Pro), GCT (Ser), GGA (Gly), GTG (Val),ATC (Ile), ATT (Ile), AAG (Lys), AAC (Asn), CAA (Gln), CAC (His), GAG(Glu), GAC (Asp), TAC (Tyr), TGC (Cys), TTC (Phe), or any combinationthereof (Murray et al., 1989). Preferred codons may differ for differenttypes of plants (Wada et al., 1990).

The modified beetle luciferase proteins or fusion proteins of theinvention may be prepared by recombinant methods or by solid phasechemical peptide synthesis methods. Such methods have been known in theart since the early 1960's (Merrifield, 1963) (See also Stewart et al.,Solid Phase Peptide Synthesis, 2 ed., Pierce Chemical Co., Rockford,Ill., pp. 11-12)) and have recently been employed in commerciallyavailable laboratory peptide design and synthesis kits (CambridgeResearch Biochemicals). Such commercially available laboratory kits havegenerally utilized the teachings of Geysen et al. (1984) and provide forsynthesizing peptides upon the tips of a multitude of rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, e.g., inverting and insertingthe rod's and pin tips into appropriate solutions, amino acids are builtinto desired peptides. In addition, a number of available FMOC peptidesynthesis systems are available. For example, assembly of a polypeptideor fragment can be carried out on a solid support using an AppliedBiosystems, Inc. Model 431A automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

IV. Fusion Partners Useful with the Modified Reporter Protein of theInvention

The polynucleotide of the invention which encodes a modified reporterprotein may be employed with other nucleic acid sequences, e.g., anative sequence such as a cDNA or one which has been manipulated invitro, e.g., to prepare N-terminal, C-terminal, or N- and C-terminalfusion proteins, e.g., a fusion with a protein encoded by a differentreporter gene including a selectable marker. Many examples of suitablefusion partners are known to the art and can be employed in the practiceof the invention.

Fusion partners include but are not limited to affinity domains or otherfunctional protein sequences, such as those having an enzymaticactivity. For example, a functional protein sequence may encode a kinasecatalytic domain (Hanks and Hunter, 1995), producing a fusion proteinthat can enzymatically add phosphate moieties to particular amino acids,or may encode a Src Homology 2 (SH2) domain (Sadowski et al., 1986;Mayer and Baltimore, 1993), producing a fusion protein that specificallybinds to phosphorylated tyrosines.

Affinity domains are generally peptide sequences that can interact witha binding partner, e.g., such as one immobilized on a solid support. DNAsequences encoding multiple consecutive single amino acids, such ashistidine, when fused to the expressed protein, may be used for one-steppurification of the recombinant protein by high affinity binding to aresin column, such as nickel sepharose. Sequences encoding peptides,such as the chitin binding domain (which binds to chitin),glutathione-S-transferase (which binds to glutathione), biotin (whichbinds to avidin and strepavidin), and the like, can also be used forfacilitating purification of the protein of interest. The affinitydomain can be separated from the protein of interest by methods wellknown in the art, including the use of inteins (protein self-splicingelements (Chong et al., 1997). Exemplary affinity domains include HisV5(HHHHH) (SEQ ID NO:1), HisX6 (HHHHHH) (SEQ ID NO:2), C-myc (EQKLISEEDL)(SEQ ID NO:3), Flag (DYKDDDDK) (SEQ ID NO:4), SteptTag (WSHPQFEK) (SEQID NO:5), hemagluttinin, e.g., HA Tag (YPYDVPDYA) (SEQ ID NO:6), GST,thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO:104),Phe-His-His-Thr (SEQ ID NO:105), chitin binding domain, S-peptide, T7peptide, SH2 domain, C-end RNA tag, WEAAAREACCRECCARA (SEQ ID NO:8),metal binding domains, e.g., zinc binding domains or calcium bindingdomains such as those from calcium-binding proteins, e.g., calmodulin,troponin C, calcineurin B, myosin light chain, recoverin, S-modulin,visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, calpainlarge-subunit, S100 proteins, parvalbumin, calbindin D_(9K), calbindinD_(28K), and calretinin, inteins, biotin, streptavidin, MyoD, Id,leucine zipper sequences, and maltose binding protein. In oneembodiment, the fusion partner is a sequence useful to purify a fusionprotein, e.g., a His or GST tag, and in one embodiment the purificationtag is fused to the N- or C-terminus of a circularly permuted reporterprotein.

Another class of fusion partners includes a protein encoded by areporter gene, including, but are not limited to, a neo gene, a β-galgene, a gus gene, a cat gene, a gpt gene, a hyg gene, a hisD gene, a blegene, a mprt gene, a bar gene, a nitrilase gene, a galactopyranosidegene, a xylosidase gene, a thymidine kinase gene, an arabinosidase gene,a mutant acetolactate synthase gene (ALS) or acetoacid synthase gene(AAS), a methotrexate-resistant dhfr gene, a dalapon dehalogenase gene,a mutated anthranilate synthase gene that confers resistance to 5-methyltryptophan (WO 97/26366), an R-locus gene, a β-lactamase gene, a xylEgene, an α-amylase gene, a tyrosinase gene, an anthozoan luciferase(luc) gene, (e.g., a Renilla reniformis luciferase gene), an aequoringene, a red fluorescent protein gene, or a green fluorescent proteingene. Included within the terms selectable or screenable marker genesare also genes which encode a “secretable marker” whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers which encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes which canbe detected by their catalytic activity. Secretable proteins fall into anumber of classes, including small, diffusible proteins detectable,e.g., by ELISA, and proteins that are inserted or trapped in the cellmembrane.

V. Vectors and Host Cells Encoding the Modified Reporter Protein orFusions Thereof

Once a desirable nucleic acid molecule encoding a modified reporterprotein or a fusion thereof is prepared, an expression cassette encodingthe modified reporter protein or a fusion protein comprising themodified reporter protein is prepared. For example, a nucleic acidmolecule comprising a nucleic acid sequence encoding a modified beetleluciferase is optionally operably linked to transcription regulatorysequences, e.g., one or more enhancers, a promoter, a transcriptiontermination sequence or a combination thereof, to form an expressioncassette. The nucleic acid molecule or expression cassette may beintroduced to a vector, e.g., a plasmid or viral vector, whichoptionally includes a selectable marker gene, and the vector introducedto a cell of interest, for example, a prokaryotic cell such as E. coli,Streptomyces spp., Bacillus spp., Staphylococcus spp. and the like, aswell as eukaryotic cells including a plant (dicot or monocot), fungus,yeast, e.g., Pichia, Saccharomyces or Schizosaccharomyces, or amammalian cell. Preferred mammalian cells include bovine, caprine,ovine, canine, feline, non-human primate, e.g., simian, and human cells.Preferred mammalian cell lines include, but are not limited to, CHO,COS, 293, Hela, CV-1, SH-SY5Y, HEK293, and NIH3T3 cells.

The expression of an encoded modified reporter protein may be controlledby any promoter capable of expression in prokaryotic cells or eukaryoticcells. Preferred prokaryotic promoters include, but are not limited to,SP6, T7, T5, tac, bla, trp, gal, lac or maltose promoters. Preferredeukaryotic promoters include, but are not limited to, constitutivepromoters, e.g., viral promoters such as CMV, SV40 and RSV promoters, aswell as regulatable promoters, e.g., an inducible or repressiblepromoter such as the tet promoter, the hsp70 promoter and a syntheticpromoter regulated by CRE. The nucleic acid molecule, expressioncassette and/or vector of the invention may be introduced to a cell byany method including, but not limited to, calcium-mediatedtransformation, electroporation, microinjection, lipofection and thelike.

VI. Exemplary Uses

The modified reporter proteins or fusions thereof are useful for anypurpose including, but not limited to, detecting the amount or presenceof a particular molecule (a biosensor), isolating a particular molecule,detecting conformational changes in a particular molecule, e.g., due tobinding, phosphorylation or ionization, detecting conditions; forinstance, pH or temperature, facilitating high or low throughputscreening, detecting protein-protein, protein-DNA or other protein-basedinteractions, or selecting or evolving biosensors. For instance, amodified reporter protein or a fusion thereof, is useful to detect e.g.,in an in vitro or cell-based assay, the amount, presence or activity ofa particular kinase (for example, by inserting a kinase site into areporter protein), RNAi (e.g., by inserting a sequence suspected ofbeing recognized by RNAi into a coding sequence for a reporter protein,then monitoring reporter activity after addition of RNAi), or protease,such as one to detect the presence of a particular viral protease, whichin turn is indicator of the presence of the virus, or an antibody; toscreen for inhibitors, e.g., protease inhibitors; to identifyrecognition sites or to detect substrate specificity, e.g., using amodified luciferase with a selected recognition sequence or a library ofmodified luciferases having a plurality of different sequences with asingle molecule of interest or a plurality (for instance, a library) ofmolecules; to select or evolve biosensors or molecules of interest,e.g., proteases; or to detect protein-protein interactions viacomplementation or binding, e.g., in an in vitro or cell-based approach.In one embodiment, a modified beetle luciferase which includes aninsertion is contacted with a random library or mutated library ofmolecules, and molecules identified which interact with the insertion.In another embodiment, a library of modified luciferases having aplurality insertions is contacted with a molecule, and modifiedluciferases which interact with the molecule identified.

The invention also provides methods to monitor the expression, locationand/or trafficking of molecules in a cell, as well as to monitor changesin microenvironments within a cell, using a modified beetle luciferaseor a fusion protein thereof. For example, in one embodiment, a modifiedbeetle luciferase comprises an internal insertion containing two domainswhich interact with each other under certain conditions. In oneembodiment, one domain in the insertion contains an amino acid which canbe phosphorylated and the other domain is a phosphoamino acid bindingdomain. In the presence of the appropriate kinase or phosphatase, thetwo domains in the insertion interact and change the conformation of themodified beetle luciferase resulting in an alteration in the detectableactivity of the modified beetle luciferase. In another embodiment, amodified beetle luciferase comprises a recognition site for a molecule,and when the molecule interacts with the recognition site, results in anincrease in activity, and so can be employed to detect or determine thepresence of amount or the other molecule.

Two-hybrid systems are extremely powerful methods for detectingprotein:protein interactions in vivo as well as identifyingresidues/domains involved in protein:protein interactions. The basis oftwo-hybrid systems is the modular domains found in some transcriptionfactors: a DNA-binding domain, which binds to a specific DNA sequence,and a transcriptional activation domain, which interacts with the basaltranscriptional machinery (Sadowski, 1988). A transcriptional activationdomain in association with a DNA-binding domain may promote the assemblyof RNA polymerase II complexes at the TATA box and increasetranscription. In the CheckMate™ Mammalian Two-Hybrid System (PromegaCorp., Madison, Wis.), the DNA-binding domain and the transcriptionalactivation domain, produced by separate plasmids, are closely associatedwhen one protein (“X”) fused to a DNA-binding domain interacts with asecond protein (“Y”) fused to a transcriptional activation domain. Inthis system, interaction between proteins X and Y results intranscription of either a reporter gene or a selectable marker gene. Inparticular, the pBIND Vector contains a yeast GAL4 DNA-binding domainupstream of a multiple cloning region, and a pACT Vector contains theherpes simplex virus VP16 activation domain upstream of a multiplecloning region. In addition, the pBIND Vector expresses the Renillareniformis luciferase. The two genes encoding the two potentiallyinteractive proteins of interest are cloned into pBIND and pACT Vectorsto generate fusion proteins with the DNA-binding domain of GAL4 and theactivation domain of VP16, respectively. The pG5luc Vector contains fiveGAL4 binding sites upstream of a minimal TATA box, which in turn, isupstream of the firefly luciferase gene (luc+). The pGAL4 and pVP16fusion constructs are transfected along with pG5luc Vector intomammalian cells. Two to three days after transfection the cells arelysed, and the amount of Renilla luciferase and firefly luciferase canbe quantitated using the Dual-Luciferase® Reporter Assay System (PromegaCat. #E1910). Interaction between the two test proteins, as GAL4 andVP16 fusion constructs, results in an increase in firefly luciferaseexpression over the negative controls. A modified beetle luciferase ofthe invention, e.g., one which is deleted at a site or region which istolerant to modification (a N-terminal fragment), is fused to a DNAbinding domain while the remainder of the beetle luciferase (theC-terminal fragment) is fused to a transcriptional activator domain.

The invention also provides methods of screening for agents (“test”agents) capable of modulating the activity of a molecule of interest.“Modulation” refers to the capacity to either enhance or inhibit afunctional property of biological activity or process (e.g., enzymeactivity); such enhancement or inhibition may be contingent on theoccurrence of a specific event, such as activation of a signaltransduction pathway, and/or may be manifest only in particular celltypes. A “modulator” refers to an agent (naturally occurring ornon-naturally occurring), such as, for example, a biologicalmacromolecule (e.g., nucleic acid, protein, non-peptide, or organicmolecule), small molecules, or an extract made from biological materialssuch as bacteria, plants, fungi, or animal (particularly mammalian)cells or tissues. Modulators are evaluated for potential activity asinhibitors or activators (directly or indirectly) of a biologicalprocess or processes (e.g., agonist, partial antagonist, partialagonist, antagonist, antineoplastic agents, cytotoxic agents, inhibitorsof neoplastic transformation or cell proliferation, cellproliferation-promoting agents, and the like) by inclusion in thescreening assays described herein. The activities (or activity) of amodulator may be known, unknown or partially known. Such modulators canbe screened using the methods of the invention. The term “test agent”refers to an agent to be tested by one or more screening method(s) ofthe invention as a putative modulator. Usually, various predeterminedconcentrations are used for screening such as 0.01 μM, 0.1 μM, 1.0 μM,and 10.0 μM. Controls can include the measurement of a signal in theabsence of the test agent, comparison to an agent known to modulate thetarget, or comparison to a sample (e. a cell, tissue or organism)before, during and/or after contacting with the test agent.

In one embodiment, the method includes screening for agents thatmodulate protease activity. For example, in one embodiment, a method ofidentifying an agent capable of modulating apoptosis is provided.Caspase family proteases have been associated with apoptosis. Thus, themethod includes contacting a sample suspected of containing acaspase-family protease with an agent suspected of modulating thecaspase activity, and a modified reporter protein having a cleavage sitecleavable by the caspase. The activity of the modified reporter proteinis detected in the sample before and after contacting with the testagent. An increase in activity after contacting with the agent isindicative of an agent that inhibits apoptosis and a decrease isindicative of an agent that activates apoptosis.

Accordingly, the invention provides a screening system useful foridentifying agents which modulate the cleavage of recognition sequencepresent in a modified reporter protein of the invention and detectingits activity. This allows one to rapidly screen for protease activitymodulators. Utilization of the screening system described hereinprovides a sensitive and rapid means to identify agents which modulate(e.g., inhibit or activate) a protease, for example, a caspase familyprotease.

A modified reporter protein of the invention is thus useful as asubstrate to study agents or conditions that modulate an interactionbetween an insertion in the modified reporter protein and a molecule ofinterest. In particular, the invention contemplates modified luciferaseproteins in which the insertion includes an amino acid sequence that isa cleavage site for an enzyme of interest. Thus, when the molecule ofinterest is a protease, the insertion comprises a peptide containing acleavage recognition sequence for the protease. A cleavage recognitionsequence for a protease is a specific amino acid sequence recognized bythe protease during proteolytic cleavage. Accordingly, the inventionprovides methods to determine the amount of a protease in a sample bycontacting the sample with a modified luciferase polypeptide of theinvention and measuring changes in luciferase activity. The modifiedluciferase protein of the invention can be used for, among other things,monitoring the activity of a protease inside a cell that expresses themodified luciferase.

The assays of the invention can be used to screen drugs to identifycompounds that alter the activity of a protease that cleaves themodified reporter protein. In one embodiment, the assay is performed ona sample in vitro containing a protease. A sample containing a knownamount of protease is mixed with a modified reporter protein of theinvention and with a test agent. The amount of the protease activity inthe sample is then determined as described above. Then the amount ofactivity per mole of protease in the presence of the test agent iscompared with the activity per mole of protease in the absence of thetest agent. A difference indicates that the test agent alters theactivity of the protease. Accordingly, the alterations may be anincrease in protease activity resulting in a decrease in modifiedreporter protein activity or a decrease in protease activitycorresponding to an increase or maintenance of modified reporter proteinactivity.

In one embodiment, the ability of an agent to alter protease activity isdetermined. In this assay, cells are conditioned or contacted with anagent suspected of modulating protease activity. The cell or cells inthe culture are lysed and protease activity measured. For example, alysed cell sample containing a known or unknown amount of protease ismixed with a modified reporter protein of the invention. The amount ofthe protease activity in the sample is then determined as above bydetermining the degree of modified reporter protein activity in acontrol or non-treated sample and the treated lysed cellular sample. Theactivity or inhibition can be calculated based on a per microgram ormilligram protein in the sample. Accordingly, the modulation in proteaseactivity includes an increase in protease activity resulting in adecrease in modified reporter protein activity or a decrease in proteaseactivity corresponding to an increase or maintenance of modifiedreporter protein activity. Typically, the difference is calibratedagainst standard measurements to yield an absolute amount of proteaseactivity. A test agent that inhibits or blocks the activity orexpression of the protease can be detected by increased modifiedreporter protein activity in treated cells compared to untreatedcontrols.

In another embodiment, the ability of an agent to alter proteaseactivity in vivo is determined. In an in vivo assay, cells transfectedwith an expression vector encoding a modified reporter protein of theinvention are exposed to different amounts of the test agent, and theeffect on reporter protein activity in a cell can be determined.Typically, the difference is calibrated against standard measurements toyield an absolute amount of protease activity. A test agent thatinhibits or blocks the activity or expression of the protease can bedetected by increased modified reporter protein activity in treatedcells compared to untreated controls.

The materials and composition for use in the assay of the invention areideally suited for the preparation of a kit. Such a kit may comprise acarrier means containing one or more container means such as vials,tubes, and the like, each of the container means comprising one of theseparate elements to be used in the method. One of the containerscomprises a modified reporter protein or polynucleotide (e.g., in theform of a vector) of the invention. A second container may contain asubstrate for the modified reporter protein.

The invention will be further described by the following non-limitingexamples.

Example I Tn5 Insertional Mutagenesis of a Click Beetle Luciferase GeneA. Transposon Insertion Reaction Target DNA Preparation

A click beetle luciferase gene (cbg69) was cloned into an E. coli T7expression vector and the resulting plasmid (pJLC1) was used as targetDNA for transposon mutagenesis reaction.

In Vitro Transposon Insertion Reaction

Reaction conditions were optimized to maximize the efficiency of thetransposon insertion while minimizing multiple insertion events. Forexample, an equimolar amount of the transposon was added to the moles oftarget DNA.

-   -   1. Prepare the transposon insertion reaction mixture by adding        in the following order:        -   1 μl 100× reaction buffer        -   0.35 μg target DNA (pJLC1) (7 μl)        -   1 μl molar equivalent transposon        -   1 μl transposase        -   10 μl total reaction volume    -   2. Incubate the reaction mixture for 2 hours at 37° C.    -   3. Stop the reaction by adding 1 μl stop solution.    -   Mix and heat for 15 minutes at 65° C.    -   The reaction mixture was stored at −20° C.

B. Selection of Transposon Insertion Clones Transformation and Recovery

The number of transposon insertion clones obtained per reaction dependson, among other factors, the transformation efficiency of the competentcells used. The greater the transformation efficiency of the competentcells, the greater the number of insertion clones obtained. A recA⁻strain of E. coli (EC100 competent cells from Epicentre) was used fortransformation.

-   -   3. Use 1 μl of the insertion reaction mixture, transform into        EC100 electrocompetent cells.    -   4. Recover the electroporated cells by adding SOC medium to the        electroporation cuvette to 1 ml final volume immediately after        electroporation. Pipette the medium/cells gently to mix.        Transfer to a tube and incubate on a 37° C. shaker for 30-60        minutes to facilitate cell outgrowth.    -   5. Plate portions of cells onto LB plates containing 50 μg/ml        kanamycin.    -   6. Grow plates overnight at 37° C.

C. Transposon Insertion Mapping

Thousands of insertion colonies were obtained. Twenty-seven insertionclones were selected and the click beetle luc gene containing Tn5transposon was PCR amplified using a primer set at the two termini ofthe cbg69 gene. The PCR products were sequenced using the same set ofprimer. The locations of the Tn5 insertion were shown to be random(FIGS. 2-3).

D. Generating a Plasmid Library of luc Gene with Transposon Insertions

Clones which had insertions in the luc gene need to be separated fromthe ones with insertions in the plasmid backbone. To do this, alltransformants were pooled and plasmid DNA was purified. The resultingplasmid DNA was digested with a pair of restriction enzymes (e.g., NdeIand EcoRI) to release a DNA fragment containing the cbg69 gene withtransposon insertions. This DNA fragment was recloned into therespective restriction enzyme sites of the E. coli T7-expression vectorfree of transposon insertions, yielding a plasmid library containing lucgene with Tn5 insertions.

E. Generating A Library of In Frame 19 Codon Insertions

Removal of Tn5 transposon

Once the plasmid library of luc gene with transposon insertions wasgenerated, the Tn5 transposon was removed by digestion with arestriction enzyme, e.g., NotI. The linearized DNA was separated fromthe DNA fragment containing Tn5 by agarose gel electrophoresis and thenpurified.

Religation and Transformation

The linearized DNA was religated using T4 DNA ligase. Successfulreligation regenerated a single restriction site, e.g., NotI, andcreated the 57 nucleotide (19 codon) insertion into one of the threereading frames. The religated DNA was transformed into EC100 cells andrecombinants were selected using an antibiotic marker present on theoriginal cloning vector (e.g., ampicillin for the control DNA).

F. Screen for Active Linker Insertion Clones

Individual linker insertion clones were used to inoculate 1 ml of LBmedium containing 100 μg/ml ampicillin and grown at 37° C. overnight.Luciferase activities were measured by mixing 100 μl of overnightculture with 100 μl Bright-Glo reagent from Promega Corp. (Madison,Wis.). Luminescence was recorded on a luminometer after 5 minutes.

G. DNA Sequencing of the active Linker Insertion Clones

Over 400 clones were screened. Linker insertion clones that hadluciferase activities >20-fold above background were selected. Thelocation of the linker insertion was determined by sequencing PCRproducts of the luc gene containing linker insertion. The positions andthe relative activities of each active linker insertion clone are shownin FIGS. 3-4.

Example II Tn-7 Insertional Mutagenesis of a Firefly Luciferase Gene

A commercial kit (GPS™-M GPS-Mutagenesis System from New England Biolabs(NEB)) was used to insert a Tn7-based transposon randomly into fireflyluciferase DNA. The major portion of this insert was then excised byrestriction enzyme digestion and religation to yield a 5 amino acidinsertion. Initially, colonies were grown and screened pre-excision forloss of luciferase activity. Plasmids in those cultures which hadluciferase activity were then excised, transformed back into cells andcolonies examined for a return of luciferase activity. Later, a moreefficient approach was used where a gel-purified luciferase fragmentcontaining the large insertion at random locations was cloned into avector and mass-excision of the vector population was performed. Here,colonies were chosen which expressed luciferase activity followingtransformation with the excised vector. Because the transposon carriedkanamycin resistance it was possible to eliminate vector molecules whichdid not contain insertions.

For the first approach, a reaction was assembled as follows:

-   -   2 μl 10×GPS buffer    -   1 μl 20 μg/ml pGPS5        -   1 μl 80 μg/ml pSP-Luc+        -   14 μl H₂0    -   20 μl        pGPS5 (NEB), which carries a kanamycin resistance gene, was the        donor plasmid, and pSP-Luc+(Promega Corp.), which has an        ampicillin resistance gene, was the acceptor. Successful        transposition resulted in the insertion of the kanamycin        resistance cassette into the acceptor plasmid. The reaction was        mixed by pipetting up and down and then 1 μl of TnsABC        Transposase was added and the reaction remixed. The reaction was        incubated for 1 hour at 37° C., heated for 10 minutes at 75° C.,        and then put on ice. 5 μl was then transformed into 100 μl high        efficiency competent E. coli JM109 (Promega Corp.). Following a        10 minute incubation on ice, the cells were subjected to a 45        second 42° C. heat shock, followed by a 2 minute incubation on        ice. 1 ml of Luria Broth (LB) was then added and the cells were        shaken at 37° C. for 1 hour. 40 μl portions were then plated on        LB agar plates containing 100 μg/ml ampicillin and 25 μg/ml        kanamycin.

The next day colonies were picked from those plates and individuallygrown in 3 ml of LB/amp/kan+0.5 mM IPTG. After overnight growth, thesecultures were assayed for luciferase activity by adding 10 μl of cultureto 100 μl of 1 mM luciferin in 100 mM sodium citrate pH 5.5 and readingstaken in a Turner 20/20 luminometer.

Plasmid was prepared from the low activity cultures (Promega Wizard PlusMinipreps kit), digested with restriction enzyme PmeI (NEB) to excisethe majority of the insert, and then religated. Typically, thesereactions were as follows:

-   -   1 μl miniprep DNA    -   1 μl 10 U/μl PmeI    -   2 μl 10× Buffer C (Promega)    -   16 μl H₂0    -   20 μl        Incubation was for 1 hour at 37° C. Reactions were then heated        at 65° C. for 20 minutes to inactivate the restriction enzyme        and the ligation reaction assembled as described below:    -   1 μl above reaction    -   3 μl 10× ligase buffer (Promega)    -   1 μl 3 U/μl T4 DNA ligase (Promega)    -   25 μl H₂0    -   30 μl        Ligations were incubated at 16° C. for at least 2 hours and then        3 μl was transformed into JM109 as described above. 501 of each        transformation was plated on either LB/amp plates or on        nitrocellulose filters overlayered on these plates. After        overnight growth at 37° C., the filters were removed and placed        on top of 1 ml of a solution of 1 mM luciferin in 100 mM sodium        citrate pH 5.5 on a slide warmer (Fisher Scientific) set to        40° C. The room was darkened and the filters observed for        luminescence. Colonies from picks observed to glow were grown up        from the LB/amp plate, plasmid was isolated and then analyzed by        restriction enzyme cutting and sequencing. Following excision of        the large kanamycin insert, a single PmeI site remains at the        site of insertion. Thus, cutting with PmeI and another        restriction enzyme allows mapping of the site of insertion.

In a second approach, a library of insertions was isolated in agel-purified luciferase fragment and cloned into a vector for excisionand expression of the protein. Specifically, transposition into pSPLuc+was accomplished as described above and then 3×5 μl was transformed into3×100 μl high efficiency JM109 as described above. 40 μl from each tubewas plated on LB/amp/kan and the cells from the remainder of this tubeas well as the other tubes was added to 50 ml LB/amp/kan and grownovernight at 37° C. The plate yielded 93 colonies corresponding to alibrary of about 7,000 different plasmids, of which about 1,400insertions were expected to be within the luciferase coding sequence.Plasmid was isolated from 8 ml of the liquid culture. Digestion of theplasmid with KpnI and EcoRI, which flank the luciferase gene, resultedin 4 fragments, corresponding to vector backbone and luciferase codingsequence, each either with or without the kanamycin insert. The band ofinterest was 3,438 bp in length and corresponded to the transposedluciferase gene fragment. About 2 μg of plasmid from the library wasdigested with KpnI and EcoRI and electrophoresed on a 1% agarose gelcontaining 1 μg/ml ethidium bromide. The 3,438 bp band was excised fromthe gel after visualization with UV illumination and purified from theagarose slice using Wizard PCR Preps (Promega Corp.). This DNA was thencloned into KpnI and EcoRI digested pGEM-3Z (Promega Corp.) followingstandard procedures. This places the luciferase gene under the controlof the Lac promoter in the vector. The majority of the kanamycin insertwas excised from the library by cutting with PmeI:

-   -   2 μl 0.25 μg/μl pGEM-3Z-luc-kan library    -   2 μl 10× Buffer C (Promega)    -   1.5 μl 10 U/μl PmeI (NEB)    -   14.5 μl H₂0    -   30 μl        This reaction was incubated at 37° C. for 1 hour, then heated        for 20 minutes at 65° C. and ligated as described below:    -   2 μl above digest    -   3 μl 10× ligase buffer (Promega)    -   1 μl 3 U/μl T4 DNA ligase (Promega)    -   24 μl H₂0    -   30 μl        The ligation reaction was incubated at 16° C. overnight and then        transformed into competent JM109 to obtain individual colonies.        By plating on plates containing only ampicillin or both        ampicillin+kanamycin it was possible to infer that approximately        90% of the transformants on ampicillin plates were sensitive to        kanamycin and thus had successfully excised the insert.        Individual colonies were cultured in 3 ml of LB+100 μg/ml        ampicillin and the cultures assayed for luciferase activity.

Results

For the first approach, about 20% of the cultures had greatly reducedluciferase activity, which is consistent with the transposon beinginserted into the luciferase coding region in the pSP-Luc+ plasmid. Forthe second approach, significant activity was observed in about 15% ofthe cultures from individual colonies. Plasmid was prepared fromcultures with activity and restriction mapping performed to identify theapproximate location of the PmeI site insert. These samples were thensubjected to standard dideoxy sequencing at the University of Iowa DNASequencing Facility. About half of the active clones contained theinsert just outside of the luciferase coding region. The remainder hadthe insert at various places within the coding region. The combinedresults from the two different methods discussed above are presentedbelow with the position of the insertion and the approximate percentactivity remaining indicated:

TABLE 1 Inserted after Amino Acid % Activity 7 10 121  5-10 233 50-75267  2 294  3 303  5-10 361 3-5 540 15 541 75

Example III Modified Click Beetle Luciferases with Modifications In theHinge Region

In order to conveniently insert various sites of interest into thepositions identified by transposon mutagenesis study, a click beetleluciferase gene (cbg69) was modified to generate two unique restrictionenzyme sites, SnaBI (TACGTA) and SalI (GTCGAC), flanking the sequenceencoding the hinge region. Specifically, two oligonucleotides:GGCTACGTAAACAATGTGGAG (SEQ ID NO:9) andGCCACTAAAGAAGCCCGTCGACGATGATGGCTGGCTC (SEQ ID NO: 18), were used tomodify the cbg69 gene using GeneEditor (Promega). The resulting clickbeetle luciferase, Cbg69ss, which has one amino acid substitution ofIle409 to Val, was shown to be twice as active as the wild-type Cbg69.The plasmid harboring cbg69ss (pJLC1ss) was used as a template togenerate other luciferases with modifications in the hinge region. Tothat end, the following pairs of oligonucleotides were synthesized:

6His-a (SEQ ID NO: 31) GTGAACCATCACCATCACCATCACAATGTGGAGGCCACTAAAGAAGCCG 6His-b (SEQ ID NO: 32)TCGACGGCTTCTTTAGTGGCCTCCACATTGTGATGGTGATGGT GATGGTTCAC FLAG-a(SEQ ID NO: 33) GTGAACGACTATAAGGACGACGACGACAAGAATGTGGAGGC CACTAAAGAAGCCGFLAG-b (SEQ ID NO: 34) TCGACGGCTTCTTTAGTGGCCTCCACATTCTTGTCGTCGTCGTCCTTATAGTCGTTCAC DEVD-a (SEQ ID NO: 35)GTGAACGACGAGGTCGACAATGTGGAGGCCACTAAAGAAGC CG DEVD-b (SEQ ID NO: 36)TCGACGGCTTCTTTAGTGGCCTCCACATTGTCGACCTCGTCGT TCAC Pka-a (SEQ ID NO: 37)GTGAACCTGCGCCGCGCCTCCCTGGGTAATGTGGAGGCCACT AAAGAAGCCG Pka-b(SEQ ID NO: 38) TCGACGGCTTCTTTAGTGGCCTCCACATTACCCAGGGAGGCGCGGCGCAGGTTCAC SARS3-a (SEQ ID NO: 39)gtaaacACTTCTGCTGTTCTGCAGAGTGGTTTTcgcAATGTGGAGG CCACTAAAGAAGCCg SARS3-b(SEQ ID NO: 40) tcgacGGCTTCTTTAGTGGCCTCCACATTgcgAAAACCACTCTGCAGAACAGCAGAAGTgtttac SARS6-a (SEQ ID NO: 41)gtaaacTCTGGTGTTACCTTCCAAGGTAAGTTCAAGAATGTGGA GGCCACTAAAGAAGCCg SARS6-b(SEQ ID NO: 42) tcgacGGCTTCTTTAGTGGCCTCCACATTCTTGAACTTACCTTGGAAGGTAACACCAGAgtttacEach oligonucleotide was phosphorylated using the following reactionconditions:

Oligonucleotide 30 pmol 10x T4 polynucleotide kinase buffer 2.5 μl 10 mMATP 2.5 μl T4 oligonucleotide kinase (1 μ/μl) 0.5 μl Water to 25 μlIncubate at 37° C. for 30 minutes and inactivate at 70° C. for 10minutes.

For each linker, a pair of phosphorylated oligonucleotides (10 μl fromabove reaction) were annealed by heating at 95° C. for 5 minutes andcooled down to 37° C. in 1 hour. Each linker was then cloned into theSnaBI and SalI sites of pJLC1ss.

Results

A. A click beetle luciferase was modified after residue 400 to contain acaspase-3 recognition site (DEVD), yielding Cbg69DEVD. Cbg69ss andCbg69DEVD were expressed in a bacterial host. The bacterial lysates weremixed with varying amounts of caspase-3 (0, 6.25, 12.5, 25, 50, 100 or200 ng) or 200 ng caspase-3 and 0.1 mM of a caspase inhibitorAc-DEVD-CHO, and luciferase activity monitored. FIG. 5A shows that ascaspase-3 concentration increased, the activity of Cbg69DEVD but notthat of Cbg69ss, decreased. Moreover, the decrease in activity was notobserved when a caspase inhibitor was present (FIG. 5B). Further, theluciferase activity decreased over time (FIG. 5C).B. SARS virus 3CL protease is a cysteine protease for SARS coronavirus,and is a potential target for an anti-SARS virus drug. Two click beetleluciferases were modified after residue 400 to contain one of two SARSprotease recognition sites (Cbg69SARS3 and Cbg69SARS6). Cbg69ss,Cbg69SARS3 and Cbg69SARS6 were produced using in vitro translationsystems such as a rabbit reticulocyte lysate and/or a wheat germ extract(Promega). The SARS protease was partially purified using a pMALpurification system from New England Biolabs. The lysates containingclick beetle luciferase were mixed with SARS protease and luciferaseactivity monitored. FIG. 6C shows that after 1 hour of incubation atroom temperature, Cbg69SARS, but not Cbg69ss, showed decreased activitywhen treated with SARS protease (about 0.3 μg) as compared to theuntreated samples.C. Modified click beetle luciferases which have various insertions sitesafter Asn400 were all active, as shown in FIGS. 6A-C. These modifiedluciferases had activities ranging from 12-64% as compared to Cbg69ss.Thus, modifications in the hinge region of click beetle luciferase canyield a modified luciferase which retains activity.

Example IV A Modified Firefly Luciferase with an Internal EnterokinaseSite

Since the 5 amino acid insertion after amino acids 233 and 541 offirefly luciferase retained the greatest fraction of enzyme activity(Example II), those sites were chosen for further analysis. TheGeneEditor™ in vitro Site-Directed Mutagenesis System (Promega Corp.)was used to perform in vitro mutagenesis to insert protease cleavagesites at these sites in order to examine the effect on luciferaseactivity after cleavage with the protease. First, the luciferase genewas cloned into the expression vector pRSET-B (Invitrogen) between theNcoI and HindIII sites using standard techniques. The luc+ gene(encoding the protein sequence shown in FIG. 7A) was excised on aNcoI-EcoRV fragment from pSPLuc+ and cloned between the NcoI and HindIIIin pRSET-B after filling in the HindIII site to create a blunt end. Thisconstruct fused luciferase amino acid sequence with an amino terminal6×His tag.

To insert an enterokinase protease cleavage site (Asp(4)Lys) intopRSET-B-luc+ after Pro233 in luc+, an oligonucleotide of the sequencePi-CCTATTTTTGGCAATCAAATCATTCCGGATGATGACGACAAGGATACTG CGATTTTAAGTGTTGTTCC(SEQ ID NO:1) was used. The plasmid template was first denatured asdescribed below:

-   -   2 μl 1 mg/ml pRSET-B-luc+    -   2 μl 2M NaOH, 2 mM EDTA    -   16 μl H₂0    -   20 μl        This mixture was incubated for 5 minutes at room temperature,        then 2 μl 2 M ammonium acetate and 75 μl 95% ethanol was added,        and the resulting mixture incubated at −20° C. for 30 minutes.        The mixture was then centrifuged in a microcentrifuge at top        speed for 5 minutes at 4° C. The pellet was then washed with 150        μl −20° C. 70% ethanol, subjected to centrifugation for 2        minutes, vacuum dried and dissolved in 100 μl TE. The mutagenic        oligonucleotide was annealed to the denatured template in the        following reaction:    -   10 μl denatured template (above)    -   1 μl 2.9 ng/μl top strand selection oligonucleotide (0.25 pmole)    -   1 μl 28 ng/μl above mutagenic oligonucleotide (1.25 pmole)    -   2 μl annealing 10× buffer    -   6 μl H₂0    -   20 μl        This annealing reaction was put in a beaker containing 200 ml of        water at 75° C. then allowed to cool in the water to 37° C. Then        the following components were added:    -   5 μl H₂0    -   3 μl 10× synthesis buffer    -   1 μl 7.7 U/μl T4 DNA polymerase    -   1 μl 3 U/μl T4 DNA ligase    -   30 μl        This reaction was incubated at 37° C. for 90 minutes after which        5 μl of the reaction was transformed into competent BMH 71-18        mutS as described in the GeneEditor™ Technical Manual. The next        day plasmid was prepared from the resulting culture and        retransformed into JM109. The resulting individual colonies were        picked, grown up, and plasmid prepared. Screening for mutants        was accomplished by digesting the plasmids with BanII and SphI        and electrophoresing the products on a 6% polyacrylamide gel        (Novex, Invitrogen) which was stained with ethidium bromide. The        digest produces a 361 bp fragment in the case of a wild-type        gene (WT) and a 376 bp fragment for the insertion mutants        containing the enterokinase site. Mutants identified in this        fashion were then confirmed by sequencing. In this experiment,        7/8 clones contained the desired insertion.

Plasmids encoding either the WT luc+ gene or the enterokinase siteinsertion were transformed into BL21(DE3)pLysS (Novagen). Transformedcultures were grown at 37° C. to an A₆₀₀ of about 0.5 and then inducedwith IPTG at 1 mM and growth continued at 37° C. for an additional 3-4hours. Cells were then pelleted and enzyme purified using MagneHis resin(Promega Corp.). Typically, 2 ml of cells were pelleted bycentrifugation for 2 minutes in a microcentrifuge. The pellet wasresuspended in 100 μl of MagneHis Wash/Binding buffer and then 10 μl of10×MLR (product #V583A) was added to lyse the cells. 5 μl of 1 U/μl RQIDNase (Promega Corp.) and 3 μl of 7 U/μl RNase One (Promega Corp.) wereadded to the lysed cells and following a 10 minute incubation on icewith occasional mixing, the lysate was spun for 5 minutes in amicrocentrifuge at 4° C. 40 μl of MagneHis resin was added to thesupernatant and the resulting mixture incubated for 5 minutes at roomtemperature with occasional mixing. The resin was then concentrated onthe tube wall by application of a magnet and washed through three cyclesof resuspension and magnetization in MagneHis Wash/Binding buffer. Theprotein was finally eluted with 100 μl of 500 mM imidazole in 100 mMHEPES pH 7.5. This procedure yielded about 5 μg of either WT or modifiedproteins.

Although the modified protein incorporated the enterokinase site, thecorresponding protease had no effect on enzyme activity and did not cutthe mutant protein after Pro233. Both WT and mutant proteins alsocontained another enterokinase site at the amino terminus which permitsremoval of the 6×His tag from the protein. Gel analysis indicated thatthis site was utilized by enterokinase in both proteins.

Another modified protein was prepared which had a Gly(3)Asp(4)LysGly(3)site inserted after Pro233 which potentially makes the enterokinase sitemore accessible. The mutagenesis was performed as above utilizing amutagenic oligonucleotide having the sequencePi-CCTATTTTTGGCAATCAAATCATTCCGGGTGGCGGTGATGATGACGACAAGGGTGGCGGTGATACTGCGATTTTAAGTGTTGTTCC (SEQ ID NO:2).

Digestion reactions were assembled as follows:

1 2 3 4 10 μl 10X EKMax 10 μl 10X EKMax 10 μl 10X EKMax 10 μl 10X EKMax2 μl WT Enzyme 2 μl WT Enzyme 1 μl Mutant Enzyme 1 μl Mutant Enzyme — 1μl 1 U/μl EKMax — 1 μl 1 U/μl EKMax 83 μl H₂0 82 μl H₂0 83 μl H₂0 82 μlH₂0 100 μl 100 μl 100 μl 100 μlEnterokinase (EKMax) and its 10× Buffer were from Invitrogen. Reactionswere incubated at room temperature and at 15 and 30 minutes, 1 μl of thereaction was added to 100 μl of Luciferase Assay Reagent (PromegaCorp.). Each sample was then read in a Turner 20/20 luminometer.This yielded the following data:

1 2 3 4  0 minutes 2517 2561 4090 3914 15 minutes 2855 2905 3460 6108 30minutes 2987 3190 3301 5717When the modified protein with the Gly(3)Asp(4)LysGly(3) site wastreated with enterokinase, luciferase activity was found to increase by50-100% (FIG. 7B). In contrast, enterokinase had no effect on theactivity of the WT enzyme. Thus, nicking of the modified luciferasebackbone did not destroy enzymatic activity. Moreover, the amino acidsequence of the insert may cause a stress on the modified protein whichis relieved by nicking with the protease, resulting in an increase inthe activity of the enzyme.

A larger insert containing an enterokinase site, i.e.,ProGlyProGly(3)Asp(4)LysGly(3)ProGlyPro, was inserted after Pro233 inLuc+. ProGlyPro was included to further increase the torsional stress onthe protein. The oligonucleotide used to create this insertion wasPi-CCTATTTTTGGCAATCAAATCATTCCGCCTGGCCCGGTGGCGGTGATGATGACGACAAGGGTGGCGGTCCTGGCCCGGATACTGCGATTTTAAGTGTTG TTCC (SEQ ID NO:3).The mutagenesis was performed as above using pRSET-B-Luc+ as thestarting plasmid. In this case, the resulting mutant plasmid wastranslated in vitro in a rabbit reticulocyte (Promega TnT® CoupledReticulocyte Lysate System) in reactions such as those below:

1 2 25 μl TnT lysate 25 μl TnT lysate 2 μl TnT reaction buffer 2 μl TnTreaction buffer 1 μl T7 RNA Polymerase 1 μl T7 RNA Polymerase 1 μl aminoacid mix 1 μl amino acid mix 1 μl 40 U/μl rRNasin 1 μl 40 U/μl rRNasin 1μl WT plasmid 1 μl Mutant plasmid 19 μl H₂0 19 μl H₂0 50 μl 50 μlReactions were incubated for 1 hour at 30° C. and then treated withenterokinase (EKMax, Invitrogen) as below:

1 2 3 4 2 μl 10 X EKMax 2 μl 10 X EKMax 2 μl 10 X EKMax 2 μl 10 X EKMax1 μl rxn 1 1 μl rxn 1 1 μl rxn 2 1 μl rxn 2 — 1 μl 1 U/μl EKMax — 1 μlEKMax 17 μl H₂0 16 μl H₂0 17 μl H₂0 16 μl H₂0 20 μl 20 μl 20 μl 20 μl1 μl was assayed in 100 μl Luciferase Assay Reagent (LAR) prior toadding the enterokinase, then at various times at room temperature afterprotease addition. The resulting data is shown in FIG. 7C. The activityof the WT enzyme was not affected by the protease whereas the modifiedenzyme was inactivated by treatment with the protease.

The effect of an enterokinase site insertion after Lys541 in Luc+ wasalso determined. In this case the oligonucleotidePi-GCAAGAAAAATCAGAGAGATCCTCATAAAGGATGATGACGACAAGGCC AAGAAGGGCGGAAAGATCGC(SEQ ID NO:4) was used with the pRSET-B-luc+ plasmid and the GeneEditorkit as described above to introduce the enterokinase site after Lys541,which is the ninth amino acid from the carboxyl end. The mutant plasmid,along with the WT as a control, was transcribed and translated inreactions similar to those described above, and then digested withenterokinase (FIG. 7D). Treatment of the modified enzyme withenterokinase reduced its activity by about 75% while the activity of theWT enzyme was not altered.

Example V A Modified Firefly Luciferase with a Deletion and HeterologousInsertion

To prepare a luciferase zymogen useful in in vitro or in vivo proteaseassays and in monitoring cellular events that are caused by or dependenton specific proteolysis, e.g., apoptosis, a firefly luciferase mutantwas constructed which had 9 amino acids inserted after Lys541 (out of550 amino acids). The 9 amino acids encoded a 5 residue enterokinaseprotease site followed by two glycines, and then 2 amino acids encodingan EcoRV site for cloning (DDDDKGGDI; SEQ ID NO:58). The vector also hadan EcoRI site outside the 3′ end of the gene which was used as a cloningsite. When the protein specified by this base construct was cut withenterokinase, the carboxy terminal 9 amino acids were removed,generating an enzyme which had about 10% the activity of the WT enzyme.A library of EcoRV and EcoRI fragments of E. coli DNA was cloned betweenthese sites in the base vector. 100 colonies were picked and assayed forluciferase activity. 7 colonies were found to have activity that wasreduced by 100-1000 fold relative to WT. The 7 colonies were culturedand plasmid prepared. The plasmids were each found to contain an insertof E. coli DNA ranging in size from about 0.2 to 3 kb. These plasmidswere translated in a TNT rabbit reticulocyte lysate and found to encodeluciferases of higher molecular weight. Enterokinase cleavage of one ofthe proteins was found to increase luciferase activity by up to 40-fold.The modified protein showing the greatest activation had a molecularweight of about 68 kD, indication that about 60 residues had beenappended to luciferase to generate the zymogen.

Example VI A Modified Firefly Luciferase which is Circularly Permuted

Plainkum et al. (2003) reported that circularly permuted forms ofribonuclease A having new N- and C-termini and a peptide linkercontaining a protease recognition site linking the original N- andC-termini had reduced ribonuclease activity due to steric occlusion ofthe active site. Plainkum et al. found that cleavage of the circularlypermuted ribonuclease A with the protease increased the activity of theprotein, presumably by removing the block to the active site.

In the case of luciferase, the N- and C-termini are separated by about40 angstroms, a distance equivalent to 5-6 amino acids. The linking theN- and C-termini of luciferase with a peptide tether may disrupt itsactivity by preventing the closure of the “lid” domain formed by thecarboxyl terminal domain of the protein. Thus, a head to tail dimer ofthe firefly luciferase luc+ gene was constructed. PCR primers weredesigned so that the upstream primer amplified beginning at Asp(234) andthe downstream primer amplified beginning at Pro(233). The upstreamprimer contained an ATG codon for a methionine just prior to Asp(234),and the downstream primer contained a stop codon. In vitro mutagenesiswas used to remove the stop codon between the original C- and N-termini,linking these termini with a sequence encoding a protease recognitionsite. For purposes of cloning the resulting PCR product, both theupstream and downstream primers also encoded a restriction enzyme site.

Methods

The head to tail luc+ dimer was constructed as follows. The vectorpSPLuc+(Promega Corp.) was digested with NcoI, the ends filled using T4DNA polymerase, and the blunt end linearized vector digested with EcoRI.To serve as the accepting vehicle, pSPLuc+ was digested with XbaI, theends filled using T4 DNA polymerase, then digested with EcoRI. Theluciferase fragment from the first digest was cloned into this vector,resulting in a head to tail arrangement of two luc+ genes in the samevector. Specifically, pSPLuc+ was digested in a reaction as follows:

-   -   1 μl 1 mg/ml pSPLuc+    -   5 μl 10× Buffer D (Promega)    -   2 μl 10 U/μl NcoI    -   42 μl H₂0    -   50 μl        The reaction was incubated for 1 hour at 37° C., heated 15        minutes at 65° C., and then chilled briefly on ice. Then 5 μl 10        mM dNTP and 1 μl 9 U/μl T4 DNA polymerase (Promega Corp.) were        added and the reaction incubated for 20 minutes at 37° C. The        reaction was purified using a Wizard Clean-Up kit (Promega        Corp.). Following elution at 65° C. in 50 μl from the Clean-Up        resin, the mixture was cooled, and the DNA was digested by        adding 5 μl 10× Buffer H (Promega Corp.) and 1 μl 12 U/μl EcoRI        (Promega Corp.). The reaction was incubated for 1 hour at 37° C.        and then heated at 65° C. for 15 minutes. The accepting vector        was then prepared as follows:    -   1 μl 1 mg/ml pSPLuc+    -   5 μl 10× Buffer D    -   1.5 μl 10 U/μl XbaI    -   42.5 μl H₂0    -   50 μl        The above reaction was incubated at 37° C. for 1 hour then        purified using the Promega Wizard Clean-Up Kit with elution in        50 μl at 65° C. The following was added to the purified DNA:    -   5 μl 10× Buffer C (Promega Corp.)    -   5 μl 10 mM dNTP    -   1 μl 9 U/μl T4 DNA Polymerase        The reaction was incubated for 20 minutes at 37° C. and then        purified as described above. 5 μl 10× Buffer H and 1 μl 12 U/μl        EcoRI was added to the eluate from the Clean-Up Resin. The        reaction was incubated for 1 hour at 37° C. and then heated at        65° C. for 15 minutes to inactivate the restriction enzyme. This        DNA was then mixed with the above digested DNA as below:    -   15 μl XbaI cut, filled EcoRI cut, heated pSPLuc+    -   25 μl NcoI cut, filled, EcoRI cut, heated pSPLuc+    -   5 μl 10× ligase buffer (Promega Corp.)    -   2 μl 3 U/μl T4 DNA ligase        After ligation overnight at 16° C., 1 μl was transformed into        high efficiency competent E. coli JM109 (Promega Corp.) and the        cells plated on LB/amp plates. Transformants were identified        which contained the correct sized plasmid. Those transformants        were expanded, plasmid isolated therefrom and the identity of        the plasmid confirmed by restriction enzyme digestion.

The head to tail dimer Luc+ DNA constructed above was used as a templatefor the PCR amplification of a permuted luciferase with a new N-terminusat Asp(234) and a new C-terminus at Pro(233). The primers used in thisamplification had the sequence:

Upstream primer = (SEQ ID NO: 109) AGCTACATATGGATACTGCGATTTTAAGTGTTGTTCDownstream primer = (SEQ ID NO: 110)AGCTAGGATCCTTACGGAATGATTTGATTGCCAAAAATAGThe amplification reaction was as follows:

-   -   5 μl 10×PfuUltra buffer (Stratagene)    -   1 μl 10 mM dNTP    -   1 μl 5 ng/μl above Luc+ dimer construct DNA    -   1 μl 100 ng/μl upstream primer    -   1 μl 100 ng/μl downstream primer    -   40 μl H₂0    -   49 μl        The reaction was mixed, overlayed with mineral oil and placed        into a PE480 thermal cycler at 95° C. After 2 minutes at this        temperature, 1 μl of 2.5 U/μl PfuUltra DNA polymerase        (Stratagene) was added and 20 cycles of 95° C. 30 seconds,        50° C. 30 seconds, 72° C. 1 minute were performed, after which        the block was brought to 4° C. The completed reaction was then        purified using Promega's Wizard PCR Preps kit and subsequent        elution from the Wizard resin in 501 of H₂0. The PCR primers        incorporated into the product have a site for NdeI (upstream        primer) or BamHI (downstream primer). The PCR product was        digested with these enzymes and cloned into the T7 expression        vector pET-3a (Novagen) as below:

1 2 5 μl 10 X Buffer D (Promega) 5 μl 10 X Buffer D (Promega) 20 μlabove PCR 1 μl 0.38 μg/μl pET-3a 1 μl 10 U/μl NdeI 1 μl 10 U/μl NdeI 1μl 10 U/μl BamHI 1 μl 10 U/μl BamHI 23 μl H₂0 42 μl H₂0 50 μl 50 μl

The above reactions were incubated at 37° C. for 1 hour, then each waspurified using the Promega Wizard Clean Up kit and DNA eluted in 50 μlof TE at 65° C. The two purified DNAs were mixed and ligated as below:

-   -   5 μl 10× ligase buffer    -   20 μl eluted 1    -   10 μl eluted 2    -   2 μl 3 U/μl T4 DNA ligase    -   13 μl H₂0    -   50 μl        The ligation reaction was incubated at 16° C. for 2 hours, then        5 μl was transformed into competent JM109 and the cells plated        on LB/amp. Colonies containing the appropriately sized plasmid        were expanded, plasmid prepared and each preparation checked for        the correct insertion by restriction digestion. Plasmid was        found containing the insertion of the PCR product and this was        used as the base vector for an in vitro mutagenesis which        eliminated the stop codon and linked the C- and N-termini at the        junction separating the two pieces of the luciferase gene.

The initial mutagenesis was performed using the Gene Editor kit fromPromega Corp. utilizing a mutagenic oligonucleotide containing arecognition site for the protease enterokinase which cleaves on thecarboxyl terminal side of Asp(4)Lys. This oligonucleotide had thesequence:

(SEQ ID NO: 7) Pi-GAAGGGCGGAAAGATCGCCGTGGATGATGACGACAAGATGGAAGACGCCAAAAACATAAAGSix colonies from the second transformation round in the mutagenesisprocedure were grown up individually and plasmid prepared therefrom.These plasmids were screened for having incorporated the mutagenicoligonucleotide by coupled transcription/translation in a TnT rabbitreticulocyte lysate (Promega Corp.). The correct mutants have fused theC- and N-termini of the luciferase domains and produce a full lengthluciferase protein. Translation reactions were performed as follows:

-   -   25 μl TnT Rabbit reticulocyte lysate    -   2 μl TnT reaction buffer    -   1 μl T7 RNA polymerase    -   1 μl complete amino acid mix    -   1 μl Fluorotect Lys tRNA    -   1 μl 40 U/μl rRNasin    -   5 μl mini prep DNA    -   14 μl    -   50 μl        The translation reactions were incubated for 60 minutes at        30° C. and then treated (or not) with enterokinase (EK) (EKMax,        Invitrogen) as below:    -   2 μl 10×EKMax buffer    -   5 μl above translation reactions    -   +/−1 μl U/μl EKMax    -   12 μl H₂0    -   20 μl        These digestions were performed at room temperature for 30        minutes then 1 μl was assayed by addition to 100 μl luciferase        assay reagent (Promega Corp.). Data collection was performed in        a Turner 20/20 luminometer. 5 μl of 4×SDS sample buffer was        added to the remainder of each reaction and the samples heated        for 2 minutes at 65° C. The samples were then electrophoresed on        a 4-20% Novex Tris-glycine gel and the gel scanned at high        sensitivity in Molecular Dynamics FluorImager. The results        indicated that the fused full-length protein was made in two of        the six clones, indicating that the mutagenesis was successful.        Moreover, the activity of the fused mutant proteins was        increased about 150-fold by treatment with enterokinase.        Furthermore, the gel showed that the protease digested the full        length protein into its pieces.

To examine the effect of EK treatment on the activity of mutantluciferases which had not been labeled by incorporation of thefluorescent lysine derivatives, translation reactions were performed asabove but the Fluorotect Lys tRNA was omitted from the reactions. Inthis case, about a 90-fold activation of luciferase activity wasobserved when the enzyme was treated with EK (FIG. 7). Followingactivation, the mutant enzyme regained about 0.5% of the WT activity.

Another mutagenesis was performed to insert a caspase-3 DEVD cleavagesite between the two luciferase domains. The Promega Gene Editor kit wasused with the following mutagenic oligonucleotide:

(SEQ ID NO: 111) Pi-GAAGGGCGGAAAGATCGCCGTGGACGAAGTTGACGGTATGGAAGACGCCAAAAACATAAAG

In this case the desired mutant was found in 5/8 clones, and screened byin vitro transcription/translation. It was found that the foldactivation by caspase-3 was higher than the fold activation previouslyobserved for enterokinase. Also, the percent of activity restored bycleavage was also greater.

In vitro translations were done in Promega TnT rabbit reticulocytelysate in reactions containing either plasmid encoding permutedluciferase containing a caspase-3 DEVD cleavage site or WT luciferase.Portions of these reactions were then digested with caspase-3 (100units, BioMol) to generate the data shown in FIGS. 9-11). The activityof the WT enzyme was not affected by the protease. In contrast, theactivity of the mutant enzyme was greatly increased by treatment withcaspase-3. The fold activation in this case was about 500-fold and theactivated enzyme had about 17% the activity of the WT.

The ability of the permuted enzyme to detect caspase-3 activity was alsoexamined in luminescent protease assays. Caspase reactions wereperformed in:

-   -   10 μl 2× Caspase buffer    -   5 μl in vitro translated proteins    -   1 μl diluted caspase-3    -   4 μl H₂0    -   20 μl        Reactions contained from between 9.6 to 2333 pg of caspase-3 and        were incubated at room temperature for 90 minutes then 1 μl was        removed and added to 100 μl luciferase assay reagent for reading        in a Turner 20/20 luminometer. FIG. 9A shows the data obtained.        Replotting the lower protease amount points (FIG. 9B) shows that        the assay is capable of detecting low picogram amounts of        caspase-3. Moreover, increasing the time of incubation from 90        minutes to overnight increased the sensitivity of the assay by        an additional 4-fold (data not shown).

The synthesis and activation of the permuted luciferases was alsoexamined in TnT Wheat Germ extracts (Promega Corp.). Reactions containedthe following:

-   -   25 μl TnT T7 WG extract    -   2 μl TnT reaction buffer    -   1 μl T7 RNA polymerase    -   1 μl amino acid mix    -   1 μl 40 U/μl rRNasin    -   5 μl 50 ng/μl luciferase plasmids    -   15 μl H₂0    -   50 μl        Reactions were incubated at 30° C. for 90 minutes then digested        with proteases as below:    -   10 μl 2× buffer (100 mM HEPES pH 7.5, 200 mM NaCl, 0.2% CHAPS, 2        mM EDTA, 20% glycerol, 20 mM DTT)    -   10 μl in vitro translation reactions    -   1 μl U/μl EKMax or 1 μl 100 U/μl Caspase-3

Protease digestions were incubated at room temperature and at varioustimes 1 μl was added to 100 μl luciferase assay reagent for reading inthe Turner 20/20 luminometer. In this experiment caspase-3 increased theactivity of the permuted caspase-luciferase by about 3000-fold to aboutone quarter that of WT, and EK increased the activity of theEK-luciferase by about 300-fold to about 1.1% of WT (FIGS. 8 and 11).Note that both inserts are the same size, DEVDG in the one case andDDDDG in the other. Thus, longer sequences may be incorporated byreplacing the three N-terminal amino acids of luciferase and the sixC-terminal amino acids, respectively, from the original termini of theprotein. This should permit a site of at least 14-15 amino acids to beincorporated between the two luciferase domains. Note that the 9residues mentioned above do not appear in the corresponding crystalstructure and thus are highly flexible and likely replaceable withoutincurring a deleterious effect on the enzyme activity.

Example VII Additional Circularly Permuted Constructs

A. PSA is a protease which cleaves Semenogelin I between Gln and Ser inthe sequence Ala-Asn-Lys-Ile-Ser-Tyr-Gln-Ser-Ser-Ser-Thr-Glu (SEQ IDNO:21). To generate a modified luciferase with a cleavage substrate forPSA, an oligonucleotide for the related 12mer peptideAla-Asn-Lys-Ala-Ser-Tyr-Gln-Ser-Ala-Ser-Thr-Glu (SEQ ID NO:22) wascloned between the XhoI and NcoI sites in the plasmid constructdescribed in Example VI. An oligonucleotide having the sequenceTCGAAGCTAACAAAGCTTCCTACCAGTCTGCGTCCACCGAAC (SEQ ID NO:23) was hybridizedto an oligonucleotide having the sequenceCATGGTTCGGTGGACGCAGACTGGTAGGAAGCTTTGTTAGCT (SEQ ID NO:24). Thehybridized oligonucleotides produce a double-stranded fragment havingXhoI and NcoI compatible ends, although the NcoI site is reformed whilethe XhoI site is destroyed. A vector was digested with XhoI and NcoI andligated to the annealed oligonucleotides, followed by transformationinto E. coli. Mini-prep DNA was prepared from individual colonies andplasmids were screened for digestion with NcoI but not with XhoI,indicating incorporation of the oligonucleotide containing the proteasesite. The desired construct was translated in vitro in either a wheatgerm (WG) translation extract or a rabbit reticulocyte lysate and theresulting protein treated with purified PSA (Sigma). Translations wereperformed. Cleavage reactions were performed as below:

The reactions were incubated at room temperature for 20 or 40 minutes. 1μl of each reaction was added to 100 μl of luciferase assay reagent(LAR) and the light output recorded in a Turner 20/20 luminometer. Thefollowing data was obtained:

20 minutes (1) 3.131 LU (2)  2061 LU (3) 0.516 LU (4) 573.1 LU 40minutes (1) 3.696 LU (2)  2149 LU (3) 0.649 LU (4) 564.6 LUThe addition of PSA resulted in substantially increased light output. At20 minutes, the fold activation of the modified luciferase was 658× forthe modified luciferase synthesized in the rabbit reticulocyte lysate,and 1,110× for the modified luciferase synthesize in the wheat germextract.B. PreScission protease is a fusion protein composed of GST (glutathioneS-transferase) and Rhinovirus 3C protease (Amersham). The protease cancleave between the Gln and Gly residues in the sequenceLeu-Glu-Val-Leu-Phe-Gln-Gly-Pro (SEQ ID NO:25). Oligonucleotidesspecifying this sequence were designed and had the sequence (top strand)TCGAGCTGGAAGTTCTGTTCCAGGGTCCGG (SEQ ID NO:26) and (bottom strand)CATGCCGGACCCTGGAACAGAACTTCCAGC (SEQ ID NO:27). The annealing of theseoligonucleotides resulted in a double-stranded fragment having XhoI andNcoI compatible ends, in which the XhoI site is retained while the NcoIsite is destroyed. As in the above example, the annealedoligonucleotides were cloned into a vector which was cut with XhoI andNcoI. To enrich for the desired clones, the ligation mix was recut withNcoI prior to transformation. The desired plasmid was selected andsubjected to in vitro translation in a rabbit reticulocyte lysate asabove. A digestion reaction was prepared as below:

The reactions were incubated at room temperature and at various times, 1μl was added to 100 μl LAR and samples read in a Turner 20/20luminometer. The following data was generated:

20 minutes (1) 0.556 LU (2)  2242 LU 40 minutes (1) 0.595 LU (2)  2500LU 60 minutes (1) 0.610 LU (2)  2447 LUActivation of the luciferase with PreScission protease occurred quicklyand resulted in a greater than 4,000 fold increase in luminescence inthe presence of the protease. C. While a high degree of activation wasobserved by proteolytic treatment of permuted luciferases synthesized ineukaryotic cell-free lysates, a much smaller degree of activation wasobserved when the unfused proteins were synthesized in E. coli.Interestingly, partial purification of the E. coli preparations producedproteins with an increased ability to be activated by protease. Toefficiently purify the circularly permuted luciferases from bacterialcells, a vector was prepared in which a circularly permuted luciferasehaving a caspase-3 site was fused to GST in the vector pGEX-6P3(Amersham). The PCR reaction contained:5 μl 10×PfuUltra buffer1 μl 10 mM dNTP1 μl 5 ng/μl caspase-3 site plasmid1 μl 100 ng/μl upstream oligonucleotide1 μl 100 ng/μl downstream oligonucleotide

-   -   40 μl H₂0    -   50 μl        The PCR was initiated by the addition of 1 μl 2.5 μ/μl PfuUltra        DNA polymerase (Stratagene) and was cycled at 95° C. for 30        seconds, 50° C. for 30 seconds, and 72° C. for 1 minute, for 20        cycles, then brought to 4° C.

The upstream oligonucleotide contains a BamH1 and has the sequenceAGCTAGGATCCGATACTGCGATTTTAAGTGTTGTTC (SEQ ID NO:28) and the downstreamoligonucleotide contains an EcoRI site and has the sequenceAGCTAGAATTCTTACGGAATGATTTGATTGCCAAAAATAG (SEQ ID NO:29). The resultingPCR product was digested with EcoR1 and BamH1 and cloned between thesesites in the vector, which results in an in-frame fusion of luciferaseto GST. The desired plasmid was identified and transformed into the E.coli strain Rosetta (Novagen). Cells were grown in LB medium and inducedby the addition of IPTG to 1 mM. The best growth conditions were foundto be an overnight induction at 25-26° C. Cells were collected and lysedby sonication. Following clearing by centrifugation, the supernatant wasapplied to a column containing immobilized glutathione and eluted with abuffer containing free glutathione. The yield of fusion protein wasabout one milligram per liter of initial culture. Activation withcaspase-3 was no less than about 1,200 fold and, depending on theconditions of the activation reaction, up to 50,000 fold (withactivation overnight on ice).

D. Three circularly permuted luciferases containing the SARS virusprotease site TSAVLQSGFR (SEQ ID NO:19) were generated: two for clickbeetle luciferase (CP1: R=Asn401 and CP2: R=Arg223) and one for afirefly (CP: R=Asp234) luciferase. CP2 has an insertion at a position inclick beetle luciferase which corresponds to position 234 in fireflyluciferase.

The circular permuted click beetle luciferases with a SARS virusprotease site were constructed as follows. A plasmid, pJLC33, whichcontains an insertion mutant cbg69SARS3 gene between NdeI and BamHIsites and a sequence encoding a SARS virus protease site between SnaBIand SalI as described above, was used as a starting vector. Thefollowing primer sets were used to amplify PCR fragments from pJLC1containing wild-type cbg69:

For CP1, CP1-a: (SEQ ID NO: 43) atgcgtcgacGTGAAACGCGAAAAGAACGTGATC and(SEQ ID NO: 44) atgcggatccttaGTTCACGTAGCCTTTAGAGACCATA; CP1-b:(SEQ ID NO: 45) atgccatatgAATGTGGAGGCCACTAAAGAAGCCATTG and(SEQ ID NO: 46) agtctacgtaGCCGCCAGCTTTTTCGAGGAG; For CP2, CP2-a:(SEQ ID NO: 47) atgcgtcgacGTGAAACGCGAAAAGAACGTGATC and (SEQ ID NO: 48)atgcggatccttaAGGGTCGAGAGCGTGGATCAAACG; CP2-b: (SEQ ID NO: 49)atgccatatgCGTGTGGGTACTCAATTGATCCC and (SEQ ID NO: 50)agtctacgtaGCCGCCAGCTTTTTCGAGGAG.

The PCR product of CP1-a (or CP2-a) was digested with SalI and BamHI,and cloned into the respective sites in pJLC33, yielding pJLC-cp1a (orpJLC-cp2a). The PCR product of CP1-b (or CP2-b) was digested with NdeIand SnaBI and cloned into the respective sites in pJLC-cp1a (orpJLC-cp2a). The resulting plasmid, pJLC47 (or pJLC48), contains thecircular permuted mutant 1 (or 2) of click beetle luciferase with theSARS virus protease site.

For the permuted firefly luciferase, the permuted vectors were modifiedto incorporate a linker with XhoI and NcoI sites separating the DNA forthe original N- and C-termini. The linker wasPi-GAGATCCTCATAAGGCCAAGAAGCTCGAGATGGTTCCATGGGCCAAAAA CATAAAGAAAGGCCCG(SEQ ID NO:20), which removes 6 amino acids from the C-terminus in thefirst domain and 3 amino acids from the N-terminus of the second domain.The SARS virus N-terminal autocleavage site is SITSAVLQSGFRKMA (SEQ IDNO:53). Oligonucleotides specifying this sequence were designed asfollows: TCGAATCCATCACCTCTGCTGTTCTGCAGTCCGGTTTCCGTAAAATGGCT C (topstrand, SEQ ID NO:51) andCATGGAGCCATTTTACGGAAACCGGACTGCAGAACAGCAGAGGTGATG GAT (bottom strand, SEQID NO:52). The annealed oligonucleotides retain the NcoI site and lackthe XhoI site. The annealed and digested oligonucleotides were clonedinto the base vector as above.

All three circular permuted luciferases with SARS virus protease sites,Cbg69CP1, Cbg69CP2 and FfCP, were produced using in vitro translationsystems such as a rabbit reticulocyte lysate and/or a wheat germ extract(Promega). The SARS virus protease was partially purified using a pMALpurification system from New England Biolabs. The lysates containingmutant luciferase were mixed with SARS virus protease and luciferaseactivity monitored. Cbg69CP2 and FfCP were activated 20-30-fold and60-200-fold, respectively (FIG. 12), whereas Cbg69CP1 was not activated(data not shown), after 1 hour of incubation with about 0.3 μg of SARSvirus protease at room temperature.

E. Activation of procaspase-3 to produce active caspase-3 is a proxy forthe induction of apotosis in living cells. To ascertain whether amodified luciferase could be used to monitor apotosis in cells, acircularly permuted luciferase containing a caspase-3 cleavage site wascloned into a mammalian expression vector under control of the CMVpromoter and introduced into Hela cells via transient transfection.Cells were then treated with the protein TRAIL to induce apoptosis viaactivation of the death receptor to form active caspase-8, which in turnactivates procaspase-3 to caspase-3. Thus, the appearance of activecaspase-3 should be accompanied by an increase in luminescence as theluciferase substrate is cleaved and activated by the enzyme.

A PCR fragment of permuted luciferase containing the caspase-3 cleavagesite was generated using primers containing sites for NheI and EcoRI andcloned into the vector pCI-neo (Promega) between these sites. Theamplification was performed as above with the upstream primerGACTAGCTAGCATGGATACTGCGATTTTAAGTGTTGTTC (SEQ ID NO:30). The resultingconstruct had an optimum Kozak sequence of the general form ANNATGG. DNAwas transfected into Hela cells using TransFast transfection reagent(Promega) and apoptosis was initiated by adding TRAIL protein (Biomol)at 1 μg/μl in DMEM+10% Cosmic Calf Serum. Some wells were transfectedwith the plasmid pGL3-control which carries the natural fireflyluciferase gene (non-permuted) under the control of the SV40 earlypromoter/enhancer. At the indicated times, 100 μl of Bright-Glo reagentwere added to the wells and luminescence recorded in an Orionluminometer (0.5 second reads).

Minutes (−)TRAIL (+)TRAIL (−)TRAIL (+)TRAIL 0 172 134 2734 2232 30 150186 3288 2448 60 164 330 2906 3198 90 294 1442  4058 3636 120 462 1508 3880 3946 150 398 940 2972 2856 pCaspase pCaspase pGL3-ctl pGL3-ctl

The data in FIG. 13 show that for cells receiving the permutedluciferase-caspase-3 plasmid, TRAIL protein induces luminescent activitybetween 1-2 hours after which luminescence fell off. This effect is notseen in wells containing medium alone. Wells containing pGL3-controlplasmid showed no difference between medium alone and medium+TRAIL. Thegeneral increase in LU seen upon changing the medium may be due to theinduction of protein synthesis, which for pGL3-control luciferase is inan active form, while for the pCI-neo-caspase plasmid, the luciferase isin an inactive form. The small increase seen in this case with mediumalone may be due to the accumulation of dead cells over the course ofthe assay, as a dead cell background is observed due to the stress ofthe transfection.

Example VIII Cell-Based Assays with Modified Luciferases

To provide a vector which encodes an intramolecular control and detectscaspase-3 activity, vectors which encoded a fusion protein of theinvention were prepared. Renilla luciferase (control) was fused toeither the N-terminus or the C-terminus of a modified click beetleluciferase containing DEVD after residue 400 (Cbg69DEVD). The linkersequence of (Gly(2)SerGly(4)SerGly(4)SerGly(2)) was placed between thetwo proteins.

To make a rLuc-linker-Cbg69DEVD fusion, a pair of oligonucleotides,atgcatatCATATGGCTTCCAAGGTGTACGACCCC (SEQ ID NO:54) andatgcATTAATgccaccggaaccgccgccaccgctaccgccgccaccgctgccCTGCTCGTTCTTCAGCACGCGCTCCACG (SEQ ID NO:55), was used to amplify a full length Renillaluciferase gene (rLuc) from plasmid pJLC6. The resulting PCR fragmentwas digested with NdeI and AseI, and cloned into the NdeI site ofpJLC23, which encodes Cbg69DEVD.

To make a Cbg69DEVD-linker-rLuc fusion, a pair of oligonucleotides,atgcatatCATATGGTGAAACGCGAAAAGAACGT (SEQ ID NO:56) andatgcATTAATgccaccggaaccgccgccaccgctaccgccgccaccgctGCCGCCAGCTTTTTCGAGGAGTTGCTTCAG (SEQ ID NO:57), was used to amplify a full lengthCbg69DEVD gene from plasmid pJLC23. The resulting PCR fragment wasdigested with NdeI and AseI, and cloned into the NdeI site of pJLC6,which contains the rLuc.

FIG. 14 shows that each fusion protein had Renilla luciferase as well asclick beetle luciferase activities.

Example IX Evaluation of Ability of Luciferase Fragments to Associateand Form Functional Luciferase

In one embodiment, the invention provides a system where two independentfragments of luciferase can complement each other to produce afunctional protein.

Materials and Methods

Three constructs were designed to evaluate the ability of N- andC-terminal fragments of luciferase to associate and form a functionalluciferase protein in vitro and in vivo (FIG. 15). The N-terminal 699nucleotides of the firefly luciferase gene (amino acids 1-233) wereamplified from pSP-luc+ (Promega Corporation) using forward primer5′ATGCGCTAGCCCGGGGATATCGCCACCATGGAAGACGCCAAAAACAT AAAG3′ (SEQ ID NO:60)and reverse primer 5′GATAAAAACCGTTAGTTTAGTAAGGCATTCCTAGGATCGA3′ (SEQ IDNO:61) under the following conditions: 95° C. for 2 minutes, 25 cyclesof 95° C. for seconds, 50° C. for 30 seconds, and 72° C. for 2 minutes,followed by 72° C. for 10 minutes on a Perkin Elmer 2400 ThermalCycler.A NheI restriction site was engineered onto the 5′ end of the forwardprimer and a BamHI restriction site was engineered onto the 5′ end ofthe reverse primer. The resultant N-terminal luciferase fragment wassubsequently cloned into the NheI and BamHI restriction sites of thepBIND vector using established techniques (Sambrook et al., 1989),yielding expression vector pJLC 62 (n luc).

Similarly, the C-terminal 951 nucleotides of the firefly luciferase gene(amino acids 234-550) were amplified from pSP-luc+ using forward primer5′ATGCGCTAGCCCGGGATATCGCCACCATGGATACTGCGATTTTAA3′ (SEQ ID NO:62) andreverse primer 5′TTGGCGCGCCGGATCCTTACACGGCGATCTTTCCGCCCTTCTTG3′ (SEQ IDNO:63) using the same PCR conditions described above for the N-terminalcloning. NheI and BamHI restriction sites were engineered into theprimers as described above for the N-terminal primers, and theC-terminal luciferase fragment was cloned into the NheI and BamHI of thepBIND vector, yielding expression vector pJLC 63 (c luc).

The whole luciferase gene (1650 nucleotides, 550 amino acids) was clonedinto the pBIND vector in the same manner as that used for the N- andC-terminal clones, using forward primer5′ATGCGCTAGCCCGGGATATCGCCACCATGGAAGACGCCAAAAACA3′ (SEQ ID NO:64) andreverse primer 5′TTGGCGCGCCGGATCCTTACACGGCGATCTTTCCGCCCTTCTTG3′ (SEQ IDNO:65) using the same PCR conditions described above. The resultantexpression vector, pJLC64 (full length FF), was used as a control forthe protein complementation experiments.

All constructs were verified for correct protein size using the TnT®Coupled Wheat Germ Extract System in conjunction with the FluoroTect™Green_(Lys) in vitro Translation Labeling System (Promega Corporation)following the manufacturer's protocol.

In vitro protein complementation experiments were performed using theTnT® Coupled Wheat Germ Extract System in conjunction with theFluoroTect™ Green_(Lys) in vitro Translation Labeling System (PromegaCorporation) following the manufacturer's protocol. After translation, 2μl of each sample were added to 100 μl of Luciferase Assay Reagent andluminescence was measured using a Veritas Luminometer.

In vivo complementation experiments were performed in Chinese HamsterOvary (CHO) and 293 human embryonic kidney tissue culture cells. Tissueculture cells, either CHO or 293 cells, were seeded into 6-well tissueculture plates, allowed to grow overnight at 37° C. and 5% CO₂, andtransfected at 80% confluency the following day. Transfection wasperformed using TransFast™ Transfection Reagent (Promega Corporation)according to the manufacturer's recommendations. Briefly, for controlreactions, 1 μg of either pJLC 62, pJLC 63, or pJLC 64 was transfected(3 μl TransFast™ Reagent/μg DNA) with 1 μg of pBIND control plasmid(original vector with no firefly luciferase gene) so that the finalconcentration for each transfection was 2 μg total DNA. For the proteincomplementation test, 1 μg of pJLC62 and 1 μg of pJLC63 were transfectedfollowing the same protocol. Twenty-four hours post-transfection, cellswere trypsinized and divided into two groups for each transfectioncondition. 250 μl of 1× Passive Lysis Buffer (Promega Corporation, PLB)was added to one group and 250 μl of 1 Phosphate Buffered Saline (PBS)was added to the other group. Groups with PLB were subjected to onefreeze thaw cycle at −80° C. to ensure lysis of the tissue culturecells, whereas the groups with PBS were not subjected to freeze thawthereby maintaining non-lysed cells. Luminescence from all groups wasmeasured using the Dual-Luciferase® Reporter Assay System according tothe manufacturer's recommendation. Basically, 20 μl from each group wasadded to a white, 96-well plate in triplicate and the assay wasperformed on a Veritas Luminometer. All firefly luciferase data wasnormalized to Renilla luciferase signal.

Results

All 3 constructs shown in FIG. 15 yielded a protein of the correct size(FIG. 16A). The activation of a circularly permuted firefly luciferaseupon protease cleavage described hereinabove suggested that fragments ofluciferase could complement and reconstitute enzyme activity. As can beseen in FIG. 16B (N- and C-fragments of luciferase in the same TnTreaction), in vitro protein complementation of the N- and C-terminalluciferase fragments yielded a functional protein when compared to thefull-length luciferase protein. Moreover, in vivo proteincomplementation occurred in both CHO and 293 tissue culture cells (FIG.17). Similar trends were seen even if the tissue culture cells were notlysed (PBS; data not shown).

Example X Detection of Non-Covalent Association of Luciferase FusionProteins in a Modulator System

In one embodiment, the invention provides a modulator system with anexogenous agent (effector A) that induces or enhances, or alternativelyinhibits, binding of two moieties, and optionally another exogenousagent (effector B) that dissociates, or alternatively enhances,respectively, binding the two moieties. For instance, such a system mayemploy rapamycin as an inducer of binding, and FK506 as a dissociator ofbinding, of FKBP and FRB which are fused to a luciferase.

A. In vitro Experiments Demonstrating a Luciferase Modulator System

Materials and Methods

A human codon optimized firefly luciferase gene (luc2.0) was amplifiedby polymerase chain reaction (PCR) from pGL4.10[luc2] (PromegaCorporation) (SEQ ID NO:66) using the forward primer5′ATGCAAGCTTGGATCCGTTTAAACGCCACCATGGATATCGCCAAAAAC ATTAAGAAGGGCCCAG3′(SEQ ID NO:67) and reverse primer5′GAGCTCGCGGCCGCCTCGAGTTATACGTAGATCTTGCCGCCCTTC3′ (SEQ ID NO:68) underthe following conditions: 95° C. for 2 minutes followed by 25 cycles of95° C. for 30 seconds, 50° C. for 30 seconds and 72° C. for 2 minutes,with a final extension of 72° C. for 10 minutes. NcoI and EcoRVrestriction endonuclease sites were engineered on the 5′ end of theforward primer to facilitate the generation of a N-terminal fusion withthe luciferase protein. SnaBI, NotI, and SacI restriction endonucleasesites were engineered on the 5′ of the reverse primer to facilitategeneration of a C-terminal fusion with the luciferase protein. Theamplified luciferase gene with additional cloning sites on the 5′ and 3′ends was cloned into a HindIII/SacI site of the Luciferase T7 ControlVector (Promega Corp., Cat No #L4821) replacing the luciferase genenormally present in the Control Vector. The resulting vector was calledpJLC 65. A general scheme for cloning into the in vitro expressionLuciferase T7 Control Vector can be seen in FIG. 18.

Several expression constructs were created using the pJLC 65 vector; aN-terminal fusion of FRB to the firefly luciferase (pJLC 66), aC-terminal fusion of FKBP to firefly luciferase (pJLC 67), and a doublefusion of FRB (N-terminus) and FKBP (C-terminus) to firefly luciferase(pJLC 68). FRB was obtained from a plasmid from Blue Heron containing asynthetic gene for FRB (CCATGGTGGCCATCCTCTGGCATGAGATGTGGCATGAAGGCCTGGAAGAGGCATCTCGTTTGTACTTTGGGGAAAGGAACGTGAAAGGCATGTTTGAGGTGCTGGAGCCCTTGCATGCTATGATGGAACGGGGCCCCCAGACTCTGAAGGAAACATCCTTTAATCAGGCCTATGGTCGAGATTTAATGGAGGCCCAAGAGTGGTGCAGGAAGTACATGAAATCAGGGAATGTCAAGGACCTCACCCAAGCCTGGGACCTCTATTATCATGTGTTCCGACGAATCTCAGGTGGC GGAGATATC; SEQ IDNO:69). FRB was cut from the Blue Heron vector using a NcoI restrictionendonuclease site on the 5′ end and an EcoRV restriction site on the 3′end, and was cloned into the N-terminus of the luciferase gene usingknown molecular biological techniques (Sambrook et al., 1989).

FKBP was obtained from a plasmid from Blue Heron containing a syntheticgene for FKBP (TACGTAGGTGGAGTGCAGGTGGAAACCATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAGAAATTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAATGACTCGAGGC GGCCGC; SEQ ID NO:70).FKBP was cut from the Blue Heron vector using SnaBI restrictionendonuclease site on the 5′ end and a NotI restriction endonuclease siteon the 3′ end of the gene so that the FKBP fragment could be cloned intothe C-terminus of the luciferase gene. The double fusion included FRBand FKBP on the N-terminus and C-terminus (respectively).

The four luciferase constructs were evaluated for correct expressedprotein size using the TnT® Coupled Wheat Germ Extract System inconjunction with the FluoroTect™ Green_(Lys) in vitro TranslationLabeling System (Promega Corporation) following the manufacturer'sprotocol. Briefly, in each of four reactions 1 μg of the appropriate DNAwas added to a 50 μl reaction including the FluoroTect™ Green_(Lys)tRNA. A sample from each reaction (5 μl) was run on a 10% NuPAGE® NovexPre-Cast Bis-Tris gel (Invitrogen Corporation) using 1× NuPAGE® MES SDSrunning buffer as described in the NuPAGE® Technical Guide (Version E,IM-1001). Gels were imaged using the FluorImager SI (MolecularDynamics).

For the in vitro assay, 5 μl from each TnT® reaction described abovewere separately added to 95 μl of 1× Passive Lysis Buffer (PromegaCorporation) with or without 0.2 μM rapamycin (BioMol). After additionof rapamycin, 10 μl from each sample were added to 100 μl of LuciferaseAssay Reagent (furnished with the TnT® System) and luminescence wasmeasured using a Turner 20/20 Luminometer (Turner BioSystems).

To study whether the interaction between FRB and FKBP could bemodulated, FK506, which is known to compete with rapamycin and inhibitthe interaction between the fusion partners, was used in in vitroexperiments. The double fusion FRB-luc2-FKBP was transcribed andtranslated as described above. After translation, 4 μl of sample wasmixed with 5 μl 2×FLICE buffer (100 mM HEPES, pH 7.5, 200 mM NaCl, 0.2%CHAPS, 2 mM EDTA, 20% glycerol, 20 mM DTT) and 1 μl rapamycin (10 nM)with varying concentrations of FK506 (Tacrolimus, Antibioticplus.com) of0, 1, 2, 5, 10, 20 and 40 nM (equivalence of 0, 0.82, 1.64, 4.1, 8.2,16.4 and 32.8 ng/ml Tacrolimus). The samples were incubated at roomtemperature for 15 minutes, after which 5 μl of sample was diluted in100 μl of Luciferase Assay Reagent and luminescence was measured on aTurner 20/20 Luminometer.

Results

Four constructs were prepared: luc2 (encoding a firefly luciferase; 550amino acids), FRB-luc2 (encoding a fusion of FRB and a fireflyluciferase; 644 amino acids), luc2-FKBP (encoding a fusion of a fireflyluciferase and FKBP; 657 amino acids), and FRB-luc2-FKBP (encoding adouble fusion; 771 amino acids). The four constructs (three controls andone double fusion) were evaluated for correct expressed protein sizeusing the TnT® Coupled Wheat Germ Extract System in conjunction with theFluoroTect™ Green_(Lys) in vitro Translation Labeling System. All fourconstructs yielded a protein of the correct size (FIG. 19).

The constructs were then used in experiments to detect an interactionbetween the two fusion partners FRB and FKBP in an in vitro system. Inthe presence of the inducer rapamycin, the two fusion partners shouldassociate resulting in a decrease in luminescence. The addition ofrapamycin resulted in a 20-fold reduction in relative luminescence withthe double fusion FRB-luc2-FKBP when compared to the control reactions(FIG. 20A). Therefore, in the presence of rapamycin, the two bindingpartners in the double fusion with luciferase associated, therebyrestricting the luciferase enzyme so that it could not interactefficiently with the luciferin substrate. Conversely, with addition ofincreasing amounts of FK506, luminescence of the double fusion reactionincreased in response to increasing amounts of FK506 (FIG. 20B).

B. Another Luciferase Modulator System Materials and Methods

Cloning was performed as described above with the following exceptions.

The red click beetle gene (cbr) was amplified out of pCBR-Basic (PromegaCorporation) using the forward primer 5′ATGCGATATCGTGAAACGCGAAAAGAACG3′(SEQ ID NO:71) and reverse primer 5′GCATAGATCTTACCGCCGGCCTTCACCAAC3′(SEQ ID NO:72). An EcoRV site was engineered into the 5′ end of theforward primer and a BglII was engineered into the 5′ end of the reverseprimer, and the corresponding amplified fragment subsequently clonedinto the corresponding sites in pJLC 68. The green click beetle gene(cbg) was amplified out of pCBG68-Basic (Promega Corporation) using theforward primer 5′ATGCGATATCGTGAAACGCGAAAAGAACG3′ (SEQ ID NO:73) and thereverse primer 5′GCATAGATCTTGCCGCCAGCTTTTTCGAGGAGTTG3′ (SEQ ID NO:74).The same restriction sites were engineered into these primers as for thered click beetle for cloning into the pJLC 68 vector. The Renillaluciferase gene (Rluc) was amplified from phRL-null (PromegaCorporation) using the forward primer5′ATGCTACGTAGCTTCCAAGGTGTACGACCCCG3′ (SEQ ID NO:75) and the reverseprimer 5′GCATAGATCTTCTGCTCGTTCTTCAGCACGCG3′ (SEQ ID NO:76). A SnaBI sitewas engineered into the 5′ end of the forward primer and a BglII sitewas engineered into the 5′ end of the reverse primer for cloning intothe pJLC 68 vector on the EcoRV (blunt end ligation with SnaBI) andBglII. The cloning of cbg, cbr, and Rluc into pJLC 68 resulted in doublefusions of the type FRB-luciferase-FKBP. Clones were verified forcorrect protein size (FIG. 21A). Double fusions were transcribed andtranslated and luminescence measured as described above, with theexception that for the rapamycin experiments only 0.2 μM rapamycin wasused.

Results

To determine whether a similar modulation of the FRB and FKBP systemthat was seen with the firefly luciferase protein could also be seenwith other species of luciferase, the firefly luciferase gene wasreplaced with two modified click beetle genes, red and green, fromPyrophorus plagiophalam, and the luciferase gene from Renillareniformis. The cloning of cbg, cbr, and Rluc into pJLC 68 resulted indouble fusions of FRB-luciferase-FKBP. Clones were verified for correctprotein size (FIG. 21A). Double fusions were transcribed and translated,and luminescence measured, as described above.

As seen in FIG. 21B, both green and red click beetle double fusionsshowed the same relative effect when rapamycin was present when comparedto the control (Luc2); luminescence decreased in response to rapamycin,whereas the Renilla luciferase did not respond to rapamycin.

C. In vivo Demonstration of a Luciferase Modulator System

Using pJLC 68 from Example X.A as a template, the fragment for theN-terminal fusion of FRB with luciferase (FRB-Luc2), C-terminal fusionof luciferase with FKBP (Luc2-FKBP), or double fusion (FRB-Luc2-FKBP)were amplified following the PCR program of 95° C. for 2 minutesfollowed by 25 cycles of 95° C. for 30 seconds, 50° C. for 30 secondsand 72° C. for 2 minutes, with a final extension of 72° C. for 10minutes. All forward primers for amplification were engineered tocontain a NheI restriction endonuclease on the 5′ end of the primer andall reverse primers were engineered to contain a BamHI restrictionendocuclease site on the 5′ end of the primer, thereby creatingamplification fragments flanked on the 5′ end by a NheI site and a BamHIsite of the 3′ end for cloning into the pBIND vector. Primers foramplification are as follows:

Luc2: Forward Primer: (SEQ ID NO: 77)ATGCGCTAGCCCGGGATATCGCCACCATGGATATCGCCAAAAAC ATTAAG Reverse Primer:(SEQ ID NO: 78) GCATGGATCCTTATACGTAGATCTTGCCG FRB-Luc2: Forward Primer:(SEQ ID NO: 79) TGCGCTAGCCCGGGATATCGCCACCATGGTGGCCATCCTCTGGCA TGAGReverse Primer: (SEQ ID NO: 80) GCATGGATCCTTATACGTAGATCTTGCCGLuc 2-FKBP: Forward Primer: (SEQ ID NO: 81)ATGCGCTAGCCCGGGATATCGCCACCATGGATATCGCCAAAAAC ATTAAG Reverse Primer:(SEQ ID NO: 82) GCATGGATCCTTATCATTCCAGTTTTAGAAGCTCCACATC FRP-Luc2-FKBP:Forward Primer: (SEQ ID NO: 83)TGCGCTAGCCCGGGATATCGCCACCATGGTGGCCATCCTCTGGCA TGAG Reverse Primer:(SEQ ID NO: 84) GCATGGATCCTTATCATTCCAGTTTTAGAAGCTCCACATC

Using the phRL-TK vector (Promega Corporation) as the source of the TKpromoter and vector backbone (FIG. 22A), the Renilla luciferase found inthe vector was removed and replaced with the FRB-Luc2-FKBP sequenceamplified from the pBIND-FRB-Luc2-FKBP to generate a NheI restrictionendonuclease on the 5′ end of the fragment and a XbaI site on the 3′ endof the fragment. The following primers generated the product forsubsequent insertion into the phRL-TK vector using established molecularbiological techniques (Sambrook et al., 1989).

TK FRP-Luc2-FKBP: Forward Primer: (SEQ ID NO: 85)TGCGCTAGCCCGGGATATCGCCACCATGGTGGCCATCCTCTGGCA TGAG Reverse Primer(SEQ ID NO: 86) GCATTCTAGATTAATTCCAGTTTTAGAAGCTCC

The in vivo response of the FRB-FKBP interaction to rapamycin wasstudied using D293 cells (a subpopulation of the parent ATCC CRL-1573HEK293 cells that were previously selected for their increased responseto cAMP stimulation). For all in vivo experiments, D293 cells wereseeded onto 96-well tissue culture plates at 5,000 cells/well prior totransfection and incubated at 37° C. and 10% CO₂ for at least 8 hours.The pBIND constructs and the TK double fusion construct were transfectedinto D293 cells using TransIT® LT1 Transfection Reagent (MirusCorporation) as described in the protocol using 0.1 μg DNA/0.3 μltransfection reagent per/well of a 96-well plate. Approximately 24 hoursafter transfection (FIGS. 24-25), 10 μl of 50 mM of Luciferin EF(Promega Corporation, endotoxin free) was added to each well and cellswere equilibrated for at least 15 minutes. An initial luminescentreading was measured from each sample (time point 0 minutes) and then 10μl of a 0.2 μM rapamycin stock diluted in OptiMEM tissue culture media(Invitrogen) was added to the rapamycin tests wells, leaving controlwells free of rapamycin. Plates were read following addition ofrapamycin and approximately every 15 minutes after the addition ofrapamycin up to one hour. For data shown in FIG. 24, FK506 was titeredinto reactions from 0-50 μM, and rapamycin was present at 1 μM.Luminescence was measured directly using the Veritas Luminometer.

Results

Rapamycin-mediated modulation of FRB-luciferase-FKBP was observed invivo (FIG. 23-25). Up to 5-fold and 2-fold decreases of luminescentsignal were observed using the TK or CMV promoter systems, respectively.Control constructs did not show a response to rapamycin (FIGS. 25 B-D).Moreover, FK506, which competes with rapamycin for binding to FKBP,counteracts the effect of rapamycin in a titratable manner (FIG. 24).

Example XI In Vitro Experiments with a C-terminal Modulated LuciferaseFusion System

In one embodiment, luciferase activity may be modulated by a fusion ateither the N- or C-terminus of luciferase. For instance, a luciferaseC-terminal fusion to calmodulin may be modulated by agents that modulatecalmodulin.

Materials and Methods

The human calmodulin gene (CaM) was amplified from vector pOTB7 (ATCC®Global Resource Center, MGC-1447) using the forward primer5′ATGCTACGTAGCTGACCAGCTGACTGAGGAGCAG3′ (SEQ ID NO:87) and reverse primer5′ATGCCTCGAGTCACTTTGCAGTCATCATCTGTAC3′ (SEQ ID NO:88) following theprogram: 95° C. for 5 minutes followed by 20 cycles of 95° C. for 30seconds, 60° C. for 30 seconds, 72° C. for 1 minute and 10 seconds. ASnaBI site was engineered onto the 5′ end of the forward primer and aXhoI site was engineered onto the 5′ end of the reverse primer. The CaMgene was cloned into the C-terminal end of the Luciferase T7 ControlVector with the Luc2 gene (as described above) on the SnaBI/XhoI sites,thereby creating the Luc2-CaM fusion construct. The fusion protein wasexpressed in vitro using the TnT® Coupled Reticulocyte Lysate System(Promega Corp.) according to the manufacturer's protocol. Luminescencewas measured on a Turner 20/20 luminometer.

To assay modulation of the luciferase protein by the attached CaMprotein, EGTA and CaCl₂ were sequentially added to the in vitro Luc2-CaMfusion protein lysate. Initially, 1 μl of the Luc2-CaM lysate from theTnT® reaction was added to 100 μl of Luciferase Assay Reagent (LAR,Promega Corp.) and 25 μl of the mixture was used to define baselineluminescence prior to addition of EGTA and CaCl₂. After initialluminescence was determined, 1 μl of a 75 mM EGTA solution (finalconcentration of 3 mM) was added to the lysate/LAR and luminescence wasdetermined. Once luminescence in response to the addition of EGTA wasdetermined, 1 μl of a 100 mM CaCl₂ solution was added to the lysate/LARand luminescence was then determined. Therefore, there were threeluminescent measurements of the Luc2-CaM fusion construct; 1) baseline,without addition of EGTA or Ca⁺², 2) after addition of EGTA, and 3)after addition of Ca⁺².

Results

The calmodulin protein undergoes large structural changes in response tocalcium and thereby provides another possibility to modulate luciferaseactivity through a C-terminal fusion. Without the presence of eitherEGTA or Ca⁺², CaM limits the interaction between luciferase and itssubstrate (FIG. 27, “sample”). However, upon addition of EGTA thislimitation is relieved (FIG. 27, “EGTA”) and luminescence increasesabout 9-fold. This increase in luminescence can be reversed by theaddition of Ca⁺² (FIG. 27, “CaCl₂”). Therefore, the conformation of CaMappeared to affect the luciferase activity in the Luc2-CaM fusion.

REFERENCES

-   Altschul et al., J. Mol. Biol., 215:403 (1990).-   Altschul et al., Nuc. Acids Res., 25:3389 (1977).-   Chong et al., Gene, 192:271 (1997).-   Corpet et al., Nucl. Acids Res., 16:1088 (1988).-   Einbond et al., FEBS Lett., 384:1 (1996).-   Geysen et al., Proc. Natl. Acad. Sci. USA, 3998 (1984).-   Hanks and Hunter, FASEB J, 9:576-595 (1995).-   Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915 (1989).-   Higgins et al., Gene, 73:237 (1988).-   Higgins et al., LABIOS, 5:157 (1989).-   Huang et al., LABIOS, 8:155 (1992).-   Ilsley et al., Cell Signaling, 14:183 (2002).-   Karlin & Altschul, Proc. Natl. Acad. Sci. USA, 90:5873 (1993).-   Lee et al., Anal. Biochem., 316:162 (2003).-   Liu et al., Gene, 237:153 (1999).-   Mayer and Baltimore, Trends Cell. Biol., 3:8 (1993).-   Merrifield, J. Am. Chem. Soc., 2149 (1963).-   Mils et al., Oncogene, 19:1257 (2000).-   Myers and Miller, LABIOS, 4:11 (1988).-   Ozawa et al, Analytical Chemistry, 73:2516 (2001).-   Paulmurugan et al., PNAS, 24:15603 (1999).-   Pearson et al., Methods Mol. Biol., 24:307 (1994).-   Plainkum et al., Nat. Struct. Biol., 10:115 (2003).-   Sadowski, et al., Mol. Cell. Bio., 6:4396 (1986).-   Sadowski et al., Nature, 335:563 (1988).-   Sala-Newby et al., Biochem J., 279:727 (1991).-   Sala-Newby et al., FEBS, 307:241 (1992).-   Sambrook et al., In: Molecular Cloning: A Laboratory Manual, Cold    Spring Harbor (1989).-   Stewart et al., Solid Phase Peptide Synthesis, 2 ed., Pierce    Chemical Co., Rockford, Ill., pp. 11-12).-   Wang et al., BBRC, 282:28 (2001).-   Waud et al, BBA, 1292:89 (1996).

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification, thisinvention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details herein may be varied considerably without departing fromthe basic principles of the invention.

1. A method to detect a molecule of interest in a cell, comprising: a)contacting a cell with a polynucleotide encoding a modified beetleluciferase, wherein the modified beetle luciferase comprises an internalinsertion of a heterologous amino acid sequence which interacts with amolecule of interest, which insertion is at a residue or in a region ina beetle luciferase sequence which is tolerant to modification, whereinactivity of the modified beetle luciferase is altered after the moleculeof interest interacts with the insertion sequence relative to theactivity before the interaction; and b) detecting the activity of themodified beetle luciferase encoded by the polynucleotide, therebydetecting the presence of the molecule in the cell.
 2. The method ofclaim 1, wherein the beetle luciferase is a firefly luciferase.
 3. Themethod of claim 1, wherein the beetle luciferase is a click beetleluciferase.
 4. The method of claim 1, wherein the modified beetleluciferase further comprises a deletion of beetle luciferase sequencesN-terminal and/or C-terminal to the insertion.
 5. The method of claim 1,wherein the modified beetle luciferase further comprises a deletion ofbeetle luciferase sequences at sequences corresponding to the N-terminusand/or C-terminus of the unmodified beetle luciferase.
 6. The method ofclaim 1, wherein the insertion is about 4 to about 50 amino acidresidues.
 7. The method of claim 1, wherein the insertion ormodification is in a region corresponding to residue 2 to 12, residue116 to 126, residue 228 to 238, residue 262 to 272, residue 289 to 308,residue 356 to 366, or residue 535 to 546 of a firefly luciferase. 8.The method of claim 1, wherein the insertion modification is in a regioncorresponding to residue 15 to 30, residue 112 to 122, residue 352 to362, residue 371 to 384, residue 393 to 414, or residue 485 to 495 of aclick beetle luciferase.
 9. The method of claim 1, wherein the insertionis in a hinge region of the luciferase.
 10. The method of claim 1,wherein detecting the activity of the modified beetle luciferase furthercomprises determining the amount of the molecule in the cell.
 11. Amethod to identify a moiety which interacts with a modified beetleluciferase, comprising: a) contacting a modified beetle luciferase witha library of compounds, wherein the modified beetle luciferase comprisesan internal insertion of a heterologous amino acid sequence, whichinsertion is at a residue or in a region which is tolerant tomodification, and wherein the activity of the modified beetle luciferaseis detectable; and b) identifying whether one or more compounds interactwith the modified beetle luciferase relative to the unmodified beetleluciferase.
 12. A method to identify a compound which interacts with aheterologous sequence in a modified beetle luciferase, comprising: a)contacting a modified beetle luciferase with one or more compounds,wherein the modified beetle luciferase comprises a first heterologoussequence comprising a domain at the N-terminus or the C-terminus of afragment of a beetle luciferase having at least 10% the activity of acorresponding full-length functional beetle luciferase, wherein thedomain noncovalently interacts with an exogenous agent, whichinteraction detectably alters luminescence of the modified beetleluciferase, wherein the heterologous sequence does not include a domainfrom the estrogen receptor, and wherein the interaction does not resultin protein complementation or protein splicing; and b) detectingluminescence, thereby identifying whether one or more compounds altersthe activity of the modified beetle luciferase.
 13. A method to identifya compound which interacts with a heterologous sequence in a modifiedbeetle luciferase, comprising: a) contacting the modified beetleluciferase with one or more compounds, wherein the modified beetleluciferase comprises a first heterologous sequence comprising a domainat the N-terminus and a second heterologous sequence comprising a domainat the C-terminus, wherein the domains of the first and secondheterologous sequences noncovalently interact, wherein the noncovalentinteraction detectably alters luminescence of the modified beetleluciferase, and wherein the noncovalent interaction is modulatable; andb) detecting luminescence, thereby identifying whether one or morecompounds alters the activity of the modified beetle luciferase.
 14. Amethod to detect in a sample the presence or amount of an exogenousagent which alters a noncovalent interaction, comprising: a) contactingthe sample with a modified beetle luciferase, wherein the modifiedbeetle luciferase comprises a first heterologous sequence comprising adomain at the N-terminus and a second heterologous sequence comprising adomain at the C-terminus, wherein the domains of the first and secondheterologous sequences noncovalently interact, wherein the noncovalentinteraction detectably alters luminescence of the modified beetleluciferase, and wherein the noncovalent interaction is modulatable; andb) detecting luminescence, thereby detecting the presence of theexogenous agent in the sample.
 15. The method of claim 14, wherein themodified beetle luciferase lacks one or more amino acids present at theN-terminus and/or C-terminus of the corresponding unmodified beetleluciferase.
 16. The method of claim 14, wherein the noncovalentinteraction is altered in the presence of a first exogenous agent. 17.The method of claim 16, wherein the alteration of the noncovalentinteraction is inhibited by a second exogenous agent.
 18. The method ofclaim 14, wherein each heterologous sequence has a different domain. 19.The method of claim 14, wherein each heterologous sequence has the samedomain.
 20. The method of claim 14, wherein the noncovalent interactioninhibits luminescence.
 21. The method of claim 14, wherein detecting theluminescence further comprises determining the amount of the exogenousagent in the sample.